Methods for inducing differentiation of undifferentiated mammalian cells into osteoblasts

ABSTRACT

The present invention relates to in vivo and in vitro methods, agents and compound screening assays for inducing differentiation of undifferentiated mammalian cells into osteoblasts, including bone formation enhancing pharmaceutical compositions, and the use thereof in treating and/or preventing a disease involving a systemic or local decrease in mean bone density in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/EP2004/014885, filedDec. 29, 2004, which claims priority to PCT/EP03/14994, filed Dec. 29,2003, both of which applications designate the USA, the disclosures ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to agents, and methods for identifyingcompounds, which agents and compounds induce the differentiation ofundifferentiated cells and/or osteoblast progenitor cells intoosteoblasts. In addition, the invention relates to compositions andmethods for the use thereof in limiting undesired bone loss in avertebrate at risk of such bone loss, in treating conditions that arecharacterized by undesired bone loss or by the need for bone growth, intreating fractures, and in treating cartilage disorders.

Bone remodeling relies on an equilibrium between an anabolic(osteogenic) and a catabolic (bone resorption) process. Bone is adynamic tissue that is continuously being destroyed (resorbed) andrebuilt, by an intricate interplay between two distinct cell lineages:bone-forming cells, known as osteoblasts and bone-resorbing cells, knownas osteoclasts.

A number of diseases are the direct result of a disturbance in thefine-tuned balance between bone resorption and bone formation. Thesediseases for the most part are skeletal diseases and inflict a largenumber of patients. Exemplary diseases include hypocalcaemia ofmalignancy, Paget's disease, inflammatory bone diseases such asrheumatoid arthritis and periodontal disease, focal osteogenesisoccurring during skeletal metastases, Crouzon's syndrome, rickets,opsismodysplasia, pycnodysostosis/Toulouse-Lautrec disease, andosteogenesis imperfecta. Of great significance are the chronicconditions of rheumatoid- and osteo-arthritis and osteoporosis,including age-related osteoporosis and osteoporosis associated withpost-menopausal hormone status. Other conditions characterized by theneed for bone growth include primary and secondary hyperparathyroidism,disuse osteoporosis, diabetes-related osteoporosis, andglucocorticoid-related osteoporosis. The single most prevalent bonedisease is osteoporosis, which affects 1 in 5 women over 50 and 1 in 20men over 50.

Other conditions that are characterized by the need to enhance boneformation include bone fractures, where it would be desirable tostimulate bone growth and to hasten and complete bone repair. Bonefractures are still treated exclusively using casts, braces, anchoringdevices and other strictly mechanical means. Other bone deficitconditions include bone segmental defects, periodontal disease,metastatic bone disease, osteolytic bone disease and conditions whereconnective tissue repair would be beneficial, such as healing orregeneration of cartilage defects or injury. To treat all of theseconditions, bone remodeling processes are required; however, in manyinstances, patients are encountered with poorly healing fractures orbone defects. Consequently, surgical intervention is often required toaccelerate the recovery. Such surgery may implant a prosthesis with orwithout bone grafting procedures. In many cases where the bone is tooporous or where previous implants failed to be incorporated into thebone, current medical practices can offer little or no help. There arecurrently no satisfactory pharmaceutical approaches to managing any ofthese conditions. While further bone deterioration associated withpost-menopausal osteoporosis has been decreased or prevented withestrogens or bisphosphonates, current therapies do not build new bone toreplace bone that has already deteriorated.

The activities of bone cells are regulated by a large number ofcytokines and growth factors, many of which have now been identified andcloned. Mundy has described the current knowledge related to thesefactors (Mundy 1996: Mundy 1993). Although there is a great deal ofinformation available on the factors which influence the breakdown andresorption of bone, information on factors which stimulate the formationof new bone is more limited.

The cascade of transcription factors and growth factors involved in thedifferentiation or progression from progenitor cell to functionalosteoclast is well established. In contrast, little is known about thefactors involved in the progression of progenitor cells intoosteoblasts. The mesenchymal progenitor or stem cells (MPCs) representthe starting point for the differentiation of both osteoclasts andosteoblasts. During embryonic development in vivo, bone formation occursthrough two distinct pathways: intramembranous and/or endochondralossification (see FIG. 1; taken from Nakashima and de Crombrugghe,(2003)). During intramembranous ossification, flat bones such as thoseof the skull or clavicles, are formed directly from condensations ofmesenchymal cells. During endochondral ossification, long bones, such aslimb bones, are formed from a cartilage intermediate formed duringmesenchymal condensation, which intermediate is invaded during furtherdevelopment by endothelial cells, osteoclasts and mesenchymal cells thatfurther differentiate into osteoblasts and osteocytes. As osteoblastsdifferentiate from precursors to mature bone-forming cells, they expressand secrete a number of enzymes and structural proteins of the bonematrix, including Type-1 collagen, osteocalcin, osteopontin and alkalinephosphatase (Stein et al 1990; Harris et al 1994). During the late stageof differentiation into osteoblasts, bone alkaline phosphatase activity(BAP) is up-regulated. Like alkaline phosphatase, osteocalcin andosteopontin, the BMPs are expressed by cultured osteoblasts as theyproliferate and differentiate.

A limited number of compounds have been identified that are able toinduce osteoblast differentiation in vitro, e.g., dexamethasone or byrecombinant human secreted proteins, e.g., BMP-2 or BMP-7 (Service,2000). BMPs are potent stimulators of bone formation in vitro and invivo, however there are disadvantages to their use as therapeutic agentsto enhance bone healing. Receptors for the bone morphogenetic proteinshave been identified in many tissues, and the BMPs themselves areexpressed in a large variety of tissues in specific temporal and spatialpatterns. This suggests that BMPs may have effects on many tissues otherthan bone, potentially limiting their usefulness as therapeutic agentswhen administered systemically.

It is therefore important to identify agents that can induce osteoblastdifferentiation starting from pluripotent bone marrow mesenchymalprogenitor cells or even from totipotent stem cells. Therefore, researchhas expanded into the identification of human secreted proteins andhuman receptor or mediator TARGETs that are involved in the specificmodulation of differentiation of osteoblasts or of other cell typesinvolved in bone homeostasis.

Reported Developments

A number of treatments have been developed and made available topatients suffering from osteoporosis and related skeletal diseases.These therapeutic approaches primarily are directed to increasing netbone formation and include: hormone replacement therapy (HRT); selectiveestrogen receptor modulators (SERMs); bisphosphonates; and calcitonin.While these treatments slow down bone resorption, they don't abolishfracturing because the lost bone is not sufficiently replenished.Fracturing will be prevented only if bone formation is sufficientlyincreased. Therefore, there is great interest in identifying osteogenicpathways that enhance bone anabolism as a basis for therapeuticintervention.

Parathyroid hormone (PTH) 1-34 is the only bone anabolic therapy on theosteoporosis therapeutic market. While PTH displays bone anaboliceffects when administered intermittently, it needs to be injected daily,and may have tumorgenic side effects, based on the observation thattumors form in animals treated with at PTH in high doses.

Bone morphogenetic proteins (BMPs) are another class of bone anabolictherapeutics, but have only been approved for niche markets. Receptorsfor the bone morphogenetic proteins have been identified in many tissuesother than bone, and BMPs themselves are expressed in a large variety oftissues in specific temporal and spatial patterns. This suggests thatBMPs may have effects on many tissues other than bone, potentiallylimiting their usefulness as therapeutic agents when administeredsystemically.

There is a clear need to identify additional targets that stimulateosteogenic differentiation and that can be used for the development ofnovel bone anabolic therapies.

The present invention relates to the relationship between the functionof selected proteins identified by the present inventors (hereinafterreferred to as “TARGETS”) and osteoblast differentiation in vertebratecells.

SUMMARY OF THE INVENTION

The present invention relates to a method for identifying compounds thatinduce differentiation of undifferentiated vertebrate cells intoosteoblasts, comprising contacting the compound with a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 377-599, 601-606, 867-1119, 1123-1133 under conditions thatallow said polypeptide to bind to the compound, and measuring acompound-polypeptide property related to the differentiation of saidcells into osteoblasts.

The present invention also relates to expression inhibitory agents,pharmaceutical compositions comprising the same, methods for the invitro production of bone tissue, and host cells expressing said agents.

Aspects of the present method include the in vitro assay of compoundsusing polypeptide of a TARGET, and cellular assays wherein TARGETinhibition is followed by observing indicators of efficacy, includingbone alkaline phosphatase secretion levels.

Another aspect of the invention is a method of treatment or preventionof a condition involving loss of bone density, in a subject suffering orsusceptible thereto, by administering a pharmaceutical compositioncomprising an effective bone formation enhancing amount of a TARGETinhibitor.

A further aspect of the present invention is a pharmaceuticalcomposition for use in said method wherein said inhibitor comprises apolynucleotide selected from the group of an antisense polynucleotide, aribozyme, and a small interfering RNA (siRNA), wherein said inhibitorcomprises a nucleic acid sequence complementary to, or engineered from,a naturally occurring polynucleotide sequence encoding a polypeptide,comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 377-418, 601, 867-1119, 1123-1133, or a fragment thereof.

Another further aspect of the present invention is a pharmaceuticalcomposition comprising a therapeutically effective boneformation-enhancing amount of a TARGET inhibitor or its pharmaceuticallyacceptable salt, hydrate, solvate, or prodrug thereof in admixture witha pharmaceutically acceptable carrier. The present polynucleotides andTARGET inhibitor compounds are also useful for the manufacturing of amedicament for the treatment of conditions involving bone density loss.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Intramembranous and endochondral ossification.

FIG. 2. Principle of the osteoblast differentiation assay.

FIG. 3. Lay-out of the 96 well knock-down control plate.

FIG. 4. Lay-out of the 384 well control plate.

FIG. 5. Performance of the knock-down control plate in the AP assay

FIG. 6. Dot plot representation of raw data for one SILENCESELECT®screening plate

FIG. 7. Profiling of target expression in MPCs (A) and knock-down ofgene expression by Ad-siRNA (B)

FIG. 8. Profiling of target expression in primary human OBs

FIG. 9. Analyzing the upregulation of BAP-mRNA versus PLAP- or IAP-mRNA

FIG. 10. Results mineralization assay

FIG. 11. Pipetting scheme used for screening the Ad-shRNAs at 3 MOIs.

DETAILED DESCRIPTION OF THE INVENTION

The following terms are used herein in accordance with the followingdefinitions:

The term “agent” means any molecule, including polypeptides,polynucleotides and small molecules.

The term “agonist” refers to a ligand that stimulates the receptor theligand binds to in the broadest sense.

The term “assay” means any process used to measure a specific propertyof a compound. A “screening assay” means a process used to characterizeor select compounds based upon their activity from a collection ofcompounds.

The term “binding affinity” is a property that describes how stronglytwo or more compounds associate with each other in a non-covalentrelationship. Binding affinities can be characterized qualitatively,(such as “strong”, “weak”, “high”, or “low”) or quantitatively (such asmeasuring the K_(D)).

The term “carrier” means a non-toxic material used in the formulation ofpharmaceutical compositions to provide a medium, bulk and/or useableform to a pharmaceutical composition. A carrier may comprise one or moreof such materials such as an excipient, stabilizer, or an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude aqueous or solid buffer ingredients including phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “complex” means the entity created when two or more compoundsbind to each other.

The term “compound” is used herein in the context of a “test compound”or a “drug candidate compound” described in connection with the assaysof the present invention. As such, these compounds comprise organic orinorganic compounds, derived synthetically or from natural sources. Thecompounds include inorganic or organic compounds such aspolynucleotides, lipids or hormone analogs that are characterized byrelatively low molecular weights. Other biopolymeric organic testcompounds include peptides comprising from about 2 to about 40 aminoacids and larger polypeptides comprising from about 40 to about 500amino acids, such as antibodies or antibody conjugates.

The term “condition” or “disease” means the overt presentation ofsymptoms (i.e., illness) or the manifestation of abnormal clinicalindicators (e.g., biochemical indicators). Alternatively, the term“disease” refers to a genetic or environmental risk of or propensity fordeveloping such symptoms or abnormal clinical indicators.

The term “contact” or “contacting” means bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

The term “effective amount” or “therapeutically effective amount” meansthat amount of a compound or agent that will elicit the biological ormedical response of a subject that is being sought by a medical doctoror other clinician. In particular, with regard to inducingundifferentiated vertebrate cells into osteoblasts, the term “effectiveamount” is intended to mean an effective differentiation-promotingamount of an compound or agent that will bring about a biologicallymeaningful increase in the levels of osteogenic markers, representativefor the process of differentiation into osteoblasts.

The term “endogenous” shall mean a material that a mammal naturallyproduces. Endogenous in reference to the term “protease”, “kinase”, orG-Protein Coupled Receptor (“GPCR”) shall mean that which is naturallyproduced by a mammal (for example, and not limitation, a human). Incontrast, the term non-endogenous in this context shall mean that whichis not naturally produced by a mammal (for example, and not limitation,a human). Both terms can be utilized to describe both “in vivo” and “invitro” systems. For example, and not a limitation, in a screeningapproach, the endogenous or non-endogenous TARGET may be in reference toan in vitro screening system. As a further example and not limitation,where the genome of a mammal has been manipulated to include anon-endogenous TARGET, screening of a candidate compound by means of anin vivo system is viable.

The term “expressible nucleic acid” means a nucleic acid coding for aproteinaceous molecule, an RNA molecule, or a DNA molecule.

The term “expression” comprises both endogenous expression andoverexpression by transduction.

The term “expression inhibitory agent” means a polynucleotide designedto interfere selectively with the transcription, translation and/orexpression of a specific polypeptide or protein normally expressedwithin a cell. More particularly, “expression inhibitory agent”comprises a DNA or RNA molecule that contains a nucleotide sequenceidentical to or complementary to at least about 17 sequentialnucleotides within the polyribonucleotide sequence coding for a specificpolypeptide or protein. Exemplary expression inhibitory moleculesinclude ribozymes, double stranded siRNA molecules, self-complementarysingle-stranded siRNA molecules, genetic antisense constructs, andsynthetic RNA antisense molecules with modified stabilized backbones.

The term “expressible nucleic acid” means a nucleic acid coding for aproteinaceous molecule, an RNA molecule, or a DNA molecule.

The term “GPCR” means a G-protein coupled receptor. Preferred GPCRscomprise those receptors identified by applicants as promotingosteogenic differentiation. Most preferred GPCRs are those identified inTables 1 and 2, including the naturally occurring transcript variantsthereof.

The term “hybridization” means any process by which a strand of nucleicacid binds with a complementary strand through base pairing. The term“hybridization complex” refers to a complex formed between two nucleicacid sequences by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(e.g., C_(0t) or R_(0t) analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed). The term “stringent conditions”refers to conditions that permit hybridization between polynucleotidesand the claimed polynucleotides. Stringent conditions can be defined bysalt concentration, the concentration of organic solvent, e.g.,formamide, temperature, and other conditions well known in the art. Inparticular, reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature canincrease stringency.

The term “inhibit” or “inhibiting”, in relationship to the term“response” means that a response is decreased or prevented in thepresence of a compound as opposed to in the absence of the compound.

The term “inhibition” refers to the reduction, down regulation of aprocess or the elimination of a stimulus for a process that results inthe absence or minimization of the expression of a protein orpolypeptide.

The term “induction” refers to the inducing, up-regulation, orstimulation of a process that results in the expression of a protein orpolypeptide.

The term “ligand” means an endogenous, naturally occurring moleculespecific for an endogenous, naturally occurring receptor.

The term “osteoblast differentiation” means the process ofdifferentiation of undifferentiated cells (progenitor cells or precursorcells) into osteoblasts and/or preosteoblasts. The term “osteogenesis”is used as a synonym in this context. “Abnormal osteoblastdifferentiation” means a situation where either too much or too littleosteoblast differentiation is occurring.

The term “pharmaceutically acceptable prodrugs” as used herein means theprodrugs of the compounds useful in the present invention, which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of patients with undue toxicity, irritation, allergicresponse commensurate with a reasonable benefit/risk ratio, andeffective for their intended use of the compounds of the invention. Theterm “prodrug” means a compound that is transformed in vivo to yield aneffective compound useful in the present invention or a pharmaceuticallyacceptable salt, hydrate or solvate thereof. The transformation mayoccur by various mechanisms, such as through hydrolysis in blood. Thecompounds bearing metabolically cleavable groups have the advantage thatthey may exhibit improved bioavailability as a result of enhancedsolubility and/or rate of absorption conferred upon the parent compoundby virtue of the presence of the metabolically cleavable group, thus,such compounds act as pro-drugs. A thorough discussion is provided inDesign of Prodrugs, H. Bundgaard, ed., Elsevier (1985); Methods inEnzymology; K. Widder et al, Ed., Academic Press, 42, 309-396 (1985); ATextbook of Drug Design and Development, Krogsgaard-Larsen and H.Bandaged, ed., Chapter 5; “Design and Applications of Prodrugs” 113-191(1991); Advanced Drug Delivery Reviews, H. Bundgard, 8, 1-38, (1992); J.Pharm. Sci., 77,285 (1988); Chem. Pharm. Bull., N. Nakeya et al, 32, 692(1984); Pro-drugs as Novel Delivery Systems, T. Higuchi and V. Stella,14 A.C.S. Symposium Series, and Bioreversible Carriers in Drug Design,E. B. Roche, ed., American Pharmaceutical Association and PergamonPress, 1987, which are incorporated herein by reference. An example ofthe prodrugs is an ester prodrug. “Ester prodrug” means a compound thatis convertible in vivo by metabolic means (e.g., by hydrolysis) to aninhibitor compound according to the present invention. For example anester prodrug of a compound containing a carboxy group may beconvertible by hydrolysis in vivo to the corresponding carboxy group.

The term “pharmaceutically acceptable salts” refers to the non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of compounds useful in thepresent invention.

The term “polynucleotide” refers to nucleic acids, such as doublestranded, or single stranded DNA and (messenger) RNA, and all types ofoligonucleotides. It also includes nucleic acids with modified backbonessuch as peptide nucleic acid (PNA), polysiloxane, and2′-O-(2-methoxy)ethylphosphorothioate. “Derivatives of a polynucleotide”means DNA-molecules, RNA-molecules, and oligonucleotides that comprise astretch or nucleic acid residues of the polynucleotide, e.g.polynucleotides that may have nucleic acid mutations as compared to thenucleic acid sequence of a naturally occurring form of thepolynucleotide. A derivative may further comprise nucleic acids withmodified backbones such as PNA, polysiloxane, and2′-O-(2-methoxy)ethyl-phosphorothioate, non-naturally occurring nucleicacid residues, or one or more nucleic acid substituents, such asmethyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-,and methanocarbanucleosides, or a reporter molecule to facilitate itsdetection. “Fragment of a polynucleotide” means oligonucleotides thatcomprise a stretch of contiguous nucleic acid residues that exhibitsubstantially a similar, but not necessarily identical, activity as thecomplete sequence.

The term “polypeptide” relates to proteins, proteinaceous molecules,fractions of proteins, peptides, oligopeptides, and enzymes (such askinases, proteases, GCPRs). “Derivatives of a polypeptide” relate tothose peptides, oligopeptides, polypeptides, proteins and enzymes thatcomprise a stretch of contiguous amino acid residues of the polypeptideand that retain the biological activity of the protein, e.g.polypeptides that have amino acid mutations compared to the amino acidsequence of a naturally-occurring form of the polypeptide. A derivativemay further comprise additional naturally occurring, altered,glycosylated, acylated or non-naturally occurring amino acid residuescompared to the amino acid sequence of a naturally occurring form of thepolypeptide. It may also contain one or more non-amino acid substituentscompared to the amino acid sequence of a naturally occurring form of thepolypeptide, for example a reporter molecule or other ligand, covalentlyor non-covalently bound to the amino acid sequence. “Fragment of apolypeptide” relates to peptides, oligopeptides, polypeptides, proteinsand enzymes that comprise a stretch of contiguous amino acid residues,and exhibit substantially similar, but not necessarily identical,functional activity as the complete sequence.

The term “SILENCESELECT® ”indicates the registered US service mark ofthe assignee of the present application, and is a registered trademarkoutside the US, and describes drug discovery services, and productsrelating to drug discovery, including arrayed libraries consisting ofshRNAs that can be used to knock-down human drugable targets.

The term “solvate” means a physical association of a compound useful inthis invention with one or more solvent molecules. This physicalassociation includes hydrogen bonding. In certain instances the solvatewill be capable of isolation, for example when one or more solventmolecules are incorporated in the crystal lattice of the crystallinesolid. “Solvate” encompasses both solution-phase and isolable solvates.Representative solvates include hydrates, ethanolates and methanolates.

The term “subject” includes humans and other mammals.

The term “treating” means an intervention performed with the intentionof preventing the development or altering the pathology of, and therebyalleviating a disorder, disease or condition, including one or moresymptoms of such disorder or condition. Accordingly, “treating” refersto both therapeutic treatment and prophylactic or preventative measures.Those in need of treating include those already with the disorder aswell as those in which the disorder is to be prevented. The related term“treatment,” as used herein, refers to the act of treating a disorder,symptom, disease or condition, as the term “treating” is defined above.

The term “undifferentiated mammalian cells” are pluripotent cells whichare in an early stage of specialization, i.e. cells which do not yethave their final function and can be induced to form almost any givencell type. In particular, these are cells which have not yetdifferentiated to the specific bone cells osteoblasts or osteoclasts.Such pluripotent cells are especially blood cells and cells present inbone marrow, as well as cells derived from adipose tissue. In addition,cells which can be differentiated into mesenchymal precursor cells arecontemplated in the present invention, such as, for example, totipotentstem cells such as embryonic stem cells.

The term “vectors” also relates to plasmids as well as to viral vectors,such as recombinant viruses, or the nucleic acid encoding therecombinant virus.

The term “vertebrate cells” means cells derived from animals havingvertera structure, including fish, avian, reptilian, amphibian,marsupial, and mammalian species. Preferred cells are derived frommammalian species, and most preferred cells are human cells. Mammaliancells include feline, canine, bovine, equine, caprine, ovine, porcinemurine, such as mice and rats, and rabbits.

Applicants' Invention Based on TARGET Relationship to OsteoblastDifferentiation

As noted above, the present invention is based on the present inventors'discovery that TARGETS are factors in the up-regulation and/or inductionof osteoblast differentiation. The term “TARGET” or “TARGETS” means theproteins identified in accordance with the present bone alkalinephosphatase assay to be involved in the induction osteoblastdifferentiation. The TARGETS are identified in Table 1 by gene name,gene symbol, genbank DNA/RNA accession number, genbank polypeptideaccession number, and their associated SEQ ID NOS. Table 1 also providesknock-down target sequences and their associated SEQ ID NOS of thepresent invention. Table 1A provides additional KD TARGET Sequences forthe TARGETS identified in Table 1.

The preferred TARGETS are identified in Table 2 by gene name, genesymbol, genbank DNA/RNA accession number, genbank polypeptide accessionnumber, and their associated SEQ ID NOS. Table 2 also providesknock-down target sequences and their associated SEQ ID NOS of thepresent invention. Table 2A identifies exemplary protein domainfragments of the preferred polypeptide TARGETS identified in Table 2.

TABLE 1 DNA/ KD RNA PP TARGET SEQ TARGET TARGET Genbank SEQ Genbank SEQTARGET Sequence ID Gene Gene DNA/RNA ID Polypeptide ID Id (5′→3′) NO.Name Symbol Accession NO. Accession NO. H24-049 GTACCTGC 1 arginineAVPR1B NM_000707 367 NP_000698 409 AGGTGCTC vasopressin AGC receptor 1BH24-225 ATGGGCTT 2 arginine AVPR1B NM_000707 367 NP_000698 409 CAACAGCCvasopressin ACC receptor 1B H24-001 CAACTTGT 3 G protein- GPR38NM_001507 607 NP_001498 867 ACCTGGGC coupled AGC receptor 38 H24-002GGTGAAGG 4 serine/threonine STK11, LKB1 NM_000455 608 NP_000446 868AGGTGCTG kinase 11 GAC (Peutz-Jeghers syndrome) H24-003 GAATCTCT 5hypothetical HSM801665, AL136697 609 AL136697's 1123 TCCGCAAG proteinMGC8407 protein ATC MGC8407 NM_024046 610 NP_076951 869 H24-004 CATGCTGT6 MAP kinase- MKNK2, SK236 348 SK236's protein 390 TTGAGAGC interactingMNK2 NM_017572 349 NP_060042 391 ATC serine/threonine NM_199054 350NP_951009 392 kinase 2 H24-005 TTTGTGCT 7 calcitonin CALCRL NM_005795611 NP_005786 870 GATTCCAT receptor-like GGC H24-006 GACGGTGT 8 caseinkinase CSNK1G1, NM_00101166 356 NP_001011664 398 TAATGATA 1, gamma 1CK1g1 (SK647) GCC NM_022048 357 NP_071331 399 H24-007 CTTCGGCA 9 myosinlight HSA247087, SK536 368 SK536's protein 410 CTCCTGAG chain kinasecaMLCK, NM_182493 369 NP_872299 411 TTC MLCK (AJ247087) H24-008 GCACAGTT10 Rho- ROCK2 NM_004850 600 NP_004841 601 TGAGAAGC associated, AGCcoiled-coil containing protein kinase 2 H24-009 ACGCAAAG 11 tRNA IPTNM_017646 612 NP_060116 871 TGGCCAGG isopentenyl- AGC transferase 1H24-010 CGATGTGC 12 protein kinase PRKCN, NM_005813 613 NP_005804 872CTTCAAGA C, nu PKD3 SK489 614 SK489's protein 1124 TTC H24-011 GTGCACGG13 membrane MPP6 NM_016447 615 NP_057531 873 ATTCAGAG protein, AGCpalmitoylated 6 H24-012; CTTCTACA 14 G protein- TYMSTR NM_006564 616NP_006555 874 H24-161 CGTCCATG coupled CTC receptor H24-013 CAACCTGC 15opsin 3 OPN3 NM_014322 617 NP_055137 875 TGGTGCTC (encephalopsin, GTCpanopsin) H24-014 CTCTCTTA 16 granzyme K GZMK NM_002104 362 NP_002095404 GATCTGGA (serine ACC protease, granzyme 3; tryptase II) H24-015AGCAGGAA 17 ubiquitin- AF073344, NM_006537 618 NP_006528 876 GGCGGACAspecific USP3 TAC protease 3 - ubiquitin specific protease 3 H24-016CTCCAGCT 18 L-fucose FUK NM_145059 619 NP_659496 877 GAGTGAGC kinase AGCH24-017 TAAACCAG 19 hypothetical MGC26954 NM_145025 620 NP_659462 878CAGAGGAG protein CTC MGC26954 H24-018 TCAGGTAG 20 coagulation F13A1NM_000129 621 NP_000120 879 TTGGTTCT factor XIII, A1 GAC polypeptideH24-019 CTGCGCCG 21 proteasome PSMB3 NM_002795 622 NP_002786 880AACAAATG (prosome, TAC macropain) subunit, beta type, 3 H24-020 TGTGGCGA22 ClpX CLPX NM_006660 623 NP_006651 881 CTTGTGCA caseinolytic CACprotease X homolog (E. coli) H24-021 TCTCTCAG 23 hypothetical C13orf6,NM_032859 353 NP_116248 395 TGTAGAAT protein FLJ14906 GCC FLJ14906H24-022 CCAGGAGG 24 HP43.8KD HP43.8KD NM_032557 624 NP_115946 882TGAAACCA protein CAC H24-023 TCAGGCGA 25 ubiquitin USP9X NM_004652 625NP_004643 883 CTACTTTA specific NM_021906 626 NP_068706 884 CTC protease9, X chromosome (fat facets-like Drosophila) H24-024 GTGTACTG 26 matrixMMP23A, NM_004659 340 NP_004650 382 GTACAAGG metallopro- MMP23BNM_006983 341 NP_008914 383 ACC teinase 23A- matrix metallopro- teinase23B H24-025 GAGCAGGT 27 HP43.8KD HP43.8KD NM_032557 627 NP_115946 885TTCCAAAG protein GAC H24-026 TCTCTCAT 28 APEX APEX NM_001641 628NP_001632 886 CAATACTG nuclease NM_080648 629 NP_542379 887 GTC(multifunctional NM_080649 630 NP_542380 888 DNA repair enzyme) H24-027TGCAGAGC 29 a disintegrin- ADAMTS19 NM_133638 631 NP_598377 889 AGACAAGTlike and GGC metalloprotease (reprolysin type) with thrombospondin type1 motif, 19 H24-028 TCATGTTG 30 a disintegrin ADAM22 NM_004194 632NP_004185 890 ACCAAGCA and NM_016351 633 NP_057435 891 AGCmetalloprotein- NM_021721 634 NP_068367 892 ase domain 22 NM_021722 635NP_068368 893 NM_021723 636 NP_068369 894 H24-029 CTATGCCA 31 LOC254378LOC254378 XM_174812 637 XP_174812 895 TCACCTTC TGC H24-030 TGTGCCGA 32similar to a LOC137491 XM_070459 638 XP_070459 896 AGGATGTA disintegrinand AGC metalloprotease domain 25 (testase 2) H24-031 CCGGGACA 33similar to bile LOC138529 XM_070951 639 XP_070951 897 TAACTAAAsalt-dependent TCC lipase oncofetal isoform H24-032 AGCAGGCT 34complement C9 NM_001737 640 NP_001728 898 ATGGGATC component 9 AACH24-033 CCACAAGG 35 xylulokinase XYLB NM_005108 641 NP_005099 899TTGCAGCA homolog (H. TTC influenzae) H24-035 GGGCTCAG 36 chaperone,CABC1, NM_020247 642 NP_064632 900 CCAGGAGA ABC1 activity ADCK3 SK609643 SK609's protein TTC of bc1 complex like (S. pombe) H24-036 CAGGTAGA37 fyn-related FRK NM_002031 644 NP_002022 901 CATGGCGG kinase CACH24-037 CTCTCCAG 38 G protein- GPR113 NM_153835 645 NP_722577 902GACACTGA coupled CTC receptor 113 (GPR113) H24-038 GCACGATT 39unc-51-like ULK1 NM_003565 646 NP_003556 903 TGGAGGTC kinase 1 (C. GCCelegans) H24-039 CGCTCTGG 40 neurotrophic NTRK1 NM_002529 647 NP_002520904 AGTCTCTC tyrosine TCC kinase, receptor, type 1 H24-040 CCCCAACT 41glycogen GSK3A NM_019884 648 NP_063937 905 ACACGGAG synthase TTC kinase3 alpha H24-041 GGACTCTC 42 phosphoinos- PIK3C2B NM_002646 376 NP_002637418 AGTTCAGC itide-3-kinase, ATC class 2, beta polypeptide H24-042CTTTGCGA 43 protein kinase, PRKR NM_002759 649 NP_002750 906 TACATGAGinterferon- CCC inducible double stranded RNA dependent H24-043 CTTCCTGA44 FUSED STK36, Fused AF200815 650 AF200815's 1125 AGACCAGGserine/threonine protein TTC kinase NM_015690 651 NP_056505 907 H24-044GAATGCTG 45 mixed lineage HSA311797, AJ311797 652 AJ311797's 1126GCAACAAG kinase 4alpha- HSA311798, protein ACC mixed lineage MLK4, SK691653 SK691's protein 1127 kinase 4beta KIAA1804 NM_032435 654 NP_115811908 H24-045 GAAGATGT 46 casein kinase CSNK1E, NM_001894 655 NP_001885909 CAACGCCC 1, epsilon CK1e NM_152221 656 NP_689407 910 ATC H24-046TTCTACGC 47 calcium- CASR NM_000388 657 NP_000379 911 ACCAGAAC sensingTCC receptor (hypocalciuric hypercalcemial, severe neonatalhyperparathy- roidism) H24-047 TGACGGTT 48 gonadotropin- GNRHR NM_000406658 NP_000397 912 GCATTTGC releasing CAC hormone receptor H24-048CCAGACCT 49 adrenergic, ADRA1B NM_000679 659 NP_000670 913 CGAGCAACalpha-1B-, TCC receptor H24-050 GGAGTGCA 50 dopamine DRD1 NM_000794 660NP_000785 914 ATCTGGTT receptor D1 TAC H24-051 CAGTCAGT 51 dopamine DRD3NM_000796 661 NP_000787 915 GCAACAGT receptor D3 NM_033658 662 NP_387507916 GTC NM_033659- 663 NP_387508 917 NM_033663 664 NP_387512 918 H24-052CGTGCTTG 52 opioid OPRD1 NM_000911 665 NP_000902 919 TCATGTTC receptor,GGC delta 1 H24-053 TGACAGGT 53 tachykinin TACR1 NM_001058 666 NP_001049920 TCCGTCTG receptor 1 GGC H24-054 GTACCTGC 54 chemokine CCR1 NM_001295342 NP_001286 384 GGCAGTTG (C—C motif) TTC receptor 1 H24-055 GACCATCA55 frizzled FZD2 NM_001466 667 NP_001457 921 CCATCCTG homolog 2 GCC(Drosophila) H24-056 GCAGGCAG 56 bombesin-like BRS3 NM_001727 668NP_001718 922 AGGACAGA receptor 3 TTC H24-057 CAAAGCCA 57 bombesin-likeBRS3 NM_001727 669 NP_001718 923 TGCCCGTA receptor 3 AGC H24-058AAGTGCCC 58 formyl peptide FPR1 NM_002029 670 NP_002020 924 TGGCCTTCreceptor 1 TTC H24-059 CTACCACA 59 frizzled FZD5 NM_003468 671 NP_003459925 AGCAGGTG homolog 5 TCC (Drosophila) H24-060; TGCGTGCT 60 smoothenedSMOH NM_005631 672 NP_005622 926 H24-168 TCTTTGTG homolog GGC(Drosophila) H24-061 CTACGTCA 61 melanocortin 5 MC5R NM_005913 673NP_005904 927 TCCTGTGC receptor CTC H24-062; TTCGGACA 62 retinal pigmentRRH NM_006583 674 NP_006574 928 H24-166 CCCACAAA epithelium- TGC derivedrhodopsin homolog H24-064; GTTGTCCT 63 olfactory OR1A2 NM_012352 364NP_036484 406 H24-164 GTTCTGAC receptor, GTC family 1, subfamily A,member 2 H24-065 GCAGGCTT 64 paired basic PACE4 NM_002570 675 NP_002561929 TCGAGTAT amino acid NM_138319 676 NP_612192 930 GGC cleavingNM_138320 677 NP_612193 931 system 4 NM_138321 678 NP_612194 932NM_138322 679 NP_612195 933 NM_138323 680 NP_612196 934 NM_138324 681NP_612197 935 NM_138325 682 NP_612198 936 H24-066 CCTCTCTG 65leucine-rich LGR7 NM_021634 683 NP_067647 937 TCAACACA repeat- TGCcontaining G protein- coupled receptor 7 H24-067 ATGACGCT 66beta-amyloid BBP NM_032027 684 NP_114416 938 ACGCAAGA binding proteinACC precursor H24-068 AGCGGGTG 67 proteasome PSMB5 NM_002797 685NP_002788 939 CTTACATT (prosome, GCC macropain) subunit, beta type, 5H24-069 GCAAGAAT 68 retinoic acid RARB NM_000965 686 NP_000956 940GCACAGAG receptor, beta NM_016152 687 NP_057236 941 AGC H24-070 TTTGTGCT69 similar to CTLA1 NM_033423 688 NP_219491 942 GACAGCTG granzyme B CTC(granzyme 2, cytotoxic T- lymphocyte- associated serine esterase 1) (H.sapiens) H24-071 CACCTGCT 70 KIAA1453 KIAA1453 NM_025090 689 NP_079366943 TTCTCAAT protein GCC H24-072 GGTTCTCT 71 fetuin B FETUB NM_014375690 NP_055190 944 GACTCGAA CAC H24-073 AGCACCTC 72 sentrin/SUMO- SENP3NM_015670 691 NP_056485 945 GCTGACAT specific TCC protease 3 H24-074CCTGGGCA 73 DKFZP586D2 DKFZP586 NM_018561 692 NP_061031 946 ACACCTGC 223protein D2223 TAC H24-075 CTTAAGGT 74 carboxypeptidase CPA6 NM_020361693 NP_065094 947 GGACCTGT A6 GGC H24-076 TTGCACAG 75 putative SPPL2ANM_032802 694 NP_116191 948 AAAGGAGG intramembrane TGC cleaving proteaseH24-077 GCCCGCAG 76 similar to LOC121302 XM_062575 695 XP_062575 949TCTTACAC protease AAC H24-078 GCTTCTGG 77 transglutam- TGM6 NM_198994696 NP_945345 950 TGGAGAAG inase 6 GAC H24-079 GTGTATGA 78liver-specific LST-3 NM_001009562 1120 NP_001009562 951 AGTGGTCC organicanion ACC transporter 3 H24-080 GAAATCTC 79 similar to LOC160131XM_090078 697 XP_090078 952 ACTGCTTC caspase 1, GAC isoform alphaprecursor; interleukin 1- beta convertase; interleukin 1-B convertingenzyme; IL1B- convertase H24-081 CACTTTAT 80 similar to LOC284964XM_209423 698 XP_209423 953 AAGCCTGC bA395L14.5 GGC (novel phosphogluco-mutase like protein) H24-082 TGGGCATC 81 LOC254378 LOC254378 XM_174812699 XP_174812 954 CTGGCTGT AAC H24-083 ATAGACTG 82 complement C9NM_001737 700 NP_001728 955 CAGAATGA component 9 GCC H24-084 CAGTGCCA 83neuron NAV2 NM_018162 701 NP_060632 956 AGAAGGAG navigator 2 NM_145117702 NP_660093 957 CCC H24-085 TGTGCTCG 84 highly charged D13S106ENM_005800 703 NP_005791 958 AAGGAGGA protein ATC D13S106E H24-086ATGTATGG 85 butyrylcho- BCHE NM_000055 704 NP_000046 959 ATTCCAGClinesterase ACC H24-087 GCTCTGCT 86 Hin-1 HSHIN1 NM_017493 705 NP_059963960 ATGTGTCA NM_199324 706 NP_955356 961 GTC H24-088 TGTGACTA 87polycystic PKDREJ NM_006071 707 NP_006062 962 AGCTGGAA kidney diseaseGAC (polycystin) and REJ (sperm receptor for egg jelly homolog, seaurchin)-like H24-089 TGCGCACA 88 protein PTPN1 NM_002827 708 NP_002818963 ATACTGGC tyrosine CAC phosphatase, non-receptor type 1 H24-090TTCCCGCT 89 glutamate GRIK4 NM_014619 709 NP_055434 964 ACCAGACCreceptor, TAC ionotropic, kainate 4 H24-091 AGCATGGT 90 potassium KCNK9NM_016601 710 NP_057685 965 CATTCACA channel, TCC subfamily K, member 9H24-092 ATGCAGGT 91 transient TRPM6 NM_017662 711 NP_060132 966 CCATATGTreceptor GAC potential cation channel, subfamily M, member 6 H24-093CCTTTCTC 92 ataxia ATR NM_001184 712 NP_001175 967 TGAACACGtelangiectasia GAC and Rad3 related H24-094 GTCAGGCT 93 p21(CDKN1A)-PAK6 NM_020168 374 NP_064553 416 GAATGAGG activated SK429 375 SK429'sprotein 417 AGC kinase 6 H24-095 CAGGTTCT 94 taste receptor, TAS1R3XM_371210 713 XP_371210 968 CCTCAAAC type 1, GGC member 3 H24-096GCATAGGA 95 similar to LOC223021 XM_167349 714 XP_167349 969 GTGGTCATtensin ATC H24-097 ACATCCTG 96 thousand and TAO1, PSK NM_016151 351NP_057235 393 CTGTCAGA one amino acid NM_004783 352 NP_004774 394 GCCprotein kinase- prostate derived STE20-like kinase PSK H24-098 CATGGAGT97 solute carrier SLC10A1 NM_003049 715 NP_003040 970 TCAGCAAG family 10ATC (sodium/bile acid cotransporter family), member 1 H24-099 GTTCTCCA98 solute carrier SLC16A3 NM_004207 366 NP_004198 408 GTGCCATT family 16GGC (monocarboxylic acid transporters), member 3 H24-100 TTCGGCCT 99organic ORCTL3 NM_004256 716 NP_004247 971 GGACGTCT cationic ATCtransporter-like 3 H24-101 GTGCATTG 100 interleukin 5 IL5 NM_000879 717NP_000870 972 GTGAAAGA (colony- GAC stimulating factor, eosinophil)H24-102 TGTGCAGG 101 transforming TGFB2 NM_003238 718 NP_003229 973ATAATTGC growth factor, TGC beta 2 H24-103 AGGTGTCA 102 tumor necrosisTNFRSF10A NM_003844 719 NP_003835 974 CTGTACAG factor receptor TCCsuperfamily, member 10a H24-104 AGTGCGCA 103 fibroblast FGF14 NM_004115720 NP_004106 975 TCTTCGGC growth factor CTC 14 H24-105 TTTGTGGA 104 Rashomolog RHEB2 NM_005614 721 NP_005605 976 CTCCTACG enriched in ATC brain2 H24-106 GCCCTGAT 105 NADPH- NR1 NM_014434 722 NP_055249 977 GTCCATCTdependent TCC FMN and FAD containing oxidoreductase H24-107 CATAGGGA 106interleukin 1 IL1F8 NM_014438 723 NP_055253 978 AGGACACT family, TGCmember 8 (eta) H24-108 CCTGGATG 107 interleukin 1 IL1F8 NM_014438 724NP_055253 979 TGAGAGAG family, AGC member 8 (eta) H24-109 AACTTGTA 108Ras association RASSF2 NM_014737 725 NP_055552 980 CTATGAAG(RalGDS/AF-6) NM_170774 726 NP_739580 981 GCC domain family 2 H24-110GTATTCTG 109 androgen- ARSDR1 NM_016026 727 NP_057110 982 TACACCCTregulated GGC short-chain dehydrogenase/ reductase 1 H24-111 TTCTCGCA110 peptidylpro- PPIL1 NM_016059 728 NP_057143 983 ATGGCCAA lylisomeraseTGC (cyclophilin)- like 1 H24-112 GAAGAACA 111 RAS, RASD1 NM_016084 365NP_057168 407 GCAGCCTG dexamethasone- GAC induced 1 H24-113 TCAGGCGG 112dicarbonyl/L- DCXR NM_016286 729 NP_057370 984 ATCTTGAC xylulose AGCreductase H24-114 TTCGGCAC 113 casein kinase CSNK2B NM_001320 730NP_001311 985 TGGTTTCC 2, beta CTC polypeptide H24-115 GACGAATA 114protein kinase, PRKAG2 NM_016203 731 NP_057287 986 TCAGCTCT AMP- GCCactivated, gamma 2 non- catalytic subunit H24-116 CCTCTCTA 115ATP-binding ABCG8 NM_022437 732 NP_071882 987 CGCCATCT casette, sub- ACCfamily G (WHITE), member 8 (sterolin 2) H24-117 TCTCTCCA 116 chromosomeC20orf121 NM_024331 733 NP_077307 988 CACAAACC 20 open TTC reading frame121 H24-118 GGTCGGGA 117 organic cation OKB1 NM_033125 734 NP_149116 989GGAGAACA transporter GTC OKB1 H24-119 GCGAATTC 118 solute carrierSLC26A8 NM_052961 735 NP_443193 990 CACCAGCA family 26, TTC member 8H24-120 TGTCCAGG 119 UDP UGT1A1 NM_000463 736 NP_000454 991 ACCTATTGglycosyltrans- AGC ferase 1 family, polypeptide A1 H24-121 TGACCATC 1203-hydroxy-3- HMGCR NM_000859 737 NP_000850 992 TGCATGAT methylglutaryl-GTC Coenzyme A reductase H24-122 GCATGGCA 121 hydroxypro- HPGD NM_000860738 NP_000851 993 TAGTTGGA staglandin TTC dehydrogenase 15-(NAD) H24-123ATGCAGGA 122 phospholipase PLCG2 NM_002661 739 NP_002652 994 CATGAACA C,gamma 2 ACC (phosphatidyl- inositol- specific) H24-124 ATGTGTGA 123thioredoxin TXNRD1 NM_182729 740 NP_877393 995 ATGTGGGT reductase 1NM_003330 741 NP_003321 996 TGC NM_182742 742 NP_877419 997 NM_182743743 NP_877420 998 H24-125 TGCAGGCT 124 UDP- B4GALT5 NM_004776 744NP_004767 999 ATTCTGTG Gal:betaGlcNAc AGC beta 1,4- galactosyltrans-ferase, polypeptide 5 H24-126 TCGGCACA 125 isocitrate IDH3B NM_174855745 NP_777280 1000 ACAATCTA dehydrogenase NM_006899 746 NP_008830 1001GAC 3 (NAD+) beta NM_174856 747 NP_777281 1002 H24-127 GACAGGTG 126sialyltransfer- SIAT4B NM_006927 748 NP_008858 1003 GACAGAGC ase 4B(beta- ATC galactosidase alpha-2,3- sialytrans- ferase) H24-128 TGTGCGAG127 HMT1 hnRNP HRMT1L3 NM_019854 343 NP_062828 385 ACCTCGAT methyltrans-TTC ferase-like 3 (S. cervisiae) H24-129 AGTTTGTG 128 UDP- B3GALT1NM_020981 749 NP_066191 1004 TAGGTATC Gal:betaGlcNAc GCC beta 1,3-galactosyltrans- ferase, polypeptide 1 H24-130 AGCATGAA 129 0-ENSG00000169, ENSG000001690 750 ENSG000001690 1128 AGAAACCC peroxisomalDHRS4, 66 66's protein TGC short-chain DHRS4L2 NM_021004 751 NP_0662841005 alcohol NM_198083 752 NP_932349 1006 dehydrogenase H24-131 GAAGATCA130 similar to PPIA, XM_292085 753 XP_292085 1007 CCATTGCT peptidylpro-LOC341457 XM_497621 1121 XP_497621 1008 GAC lylisomerase A LOC126170,XM_371409 754 XP_371409 1009 (cyclophilin A)- LOC388817, NM_203431 755NP_982255 1010 similar to Pep- LOC402102, XM_497870 756 XP_497870 1011tidyl-prolyl LOC440892, XM_496579 757 XP_496579 1012 cis-transLOC439953, XM_495800 758 XP_495800 1013 isomerase A LOC343384, NM_203430759 NP_982254 1015 (PPIase) OAS2, XM_291544 760 XP_291544 1016(Rotamase) LOC390299, NM_178230 761 NP_839944 1017 (Cyclophilin A)LOC388687, XM_372452 762 XP_372452 1018 (Cyclosporin LOC388686,XM_371304 763 XP_371304 1020 A-binding LOC128192, XM_371302 1122XP_371302 1021 protein) (SP18) LOC392352 XM_060887 764 XP_060887 1022XM_373301 765 XP_373301 1023 NM_021130 766 NP_066953 1024 767 H24-132GCATGAAT 131 similar to PPIA, XM_497621 1121 XP_497621 1025 ATTGTGGApeptidylpro- LOC126170, XM_371409 768 XP_371409 1026 GGC lylisomerase ALOC388817, NM_203431 769 NP_982255 1027 (cyclophilin A)- LOC402102,XM_497870 770 XP_497870 1028 similar to Pep- LOC391062, XM_372785 771XP_372785 1029 tidyl-prolyl PPIA, NM_203430 772 NP_982254- 1030cis-trans LOC390299, XM_372452 773 XP_372452 1031 isomerase A LOC391352,XM_372916 774 XP_372916 1032 (PPIase) LOC128192 XM_060887 775 XP_0608871033 (Rotamase) NM_021130 776 NP_066953 1034 (Cyclophilin A)(Cyclosporin A-binding protein) (SP18) H24-133 TGCAGGCA 132 3-oxoacidCoA OXCT2 NM_022120 777 NP_071403 1035 AGCAGACG transferase 2 GTCH24-134 TGACGCAG 133 gycosyltrans- LOC83468 NM_031302 778 NP_112592 1036ATGATGAA ferase TCC H24-135 TGGCGCCA 134 histone HDAC10 NM_032019 779NP_114408 1037 TGTCAGAG deacetylase 10 TGC H24-136 CTTATTGT 135 similarto LOC170327 XM_093255 780 XP_093255 1038 TCACATTG cytochrome P- GCC 450H24-137 TGGCACCT 136 similar to LOC166624 XM_093980 781 XP_093980 1039ATGAGAGG UDP- ATC glucuronosyl- transferase H24-138 TCAGGTGT 137interleukin-1 IRAK2 NM_001570 344 NP_001561 386 CCCATTCC receptor- SK180345 SK180's protein 387 AGC associated kinase 2 H24-139 CTCAGAGG 138IL2-inducible ITK NM_005546 782 NP_005537 1040 TGGTGGAA T-cell kinaseGAC H24-140 TGCAGGAG 139 cytochrome AF182273, AF182273 783 AF182273's1129 GAAATTGA P450, CYP3A3, protein TGC subfamily IIIA CYP3A4 NM_000776784 NP_000767 1041 (niphedipine NM_017460 785 NP_059488 1042 oxidase),polypeptide 3- cytochrome P450, subfamily IIIA (niphedipine oxidase),polypeptide 4 H24-141 GAGTCCAG 140 cytochrome HUMCYPIIF, J02906 786J02906's protein 1130 CCTTCATG P450, CYP2F1 NM_000774 787 NP_000765 1043CCC subfamily IIF, polypeptide 1 H24-142 GTCCAGCT 141 glutamate GRIN2ANM_000833 339 NP_000824 381 GAAGAAGA receptor, TCC ionotropic, N- methylD- aspartate 2A H24-143 TTCGGCAC 142 hypothetical FLJ22955 NM_024819 359NP_079095 401 TGAGGTCT protein TGC FLJ22955 H24-144 TTGCACAC 143hypothetical FLJ22955 NM_024819 866 NP_079095 1044 TGAGCTGG protein AGCFLJ22955 H24-145 CCTGCTCT 144 tumor TEM5 NM_032777 788 NP_116166 1045TGAGCAAT endothelial AAC marker 5 precursor H24-146 TGTCCAGA 145 similarto LOC126537 XM_497611 789 XP_497611 1046 CCACATGG cytochrome AGC P450,subfamily IVF, polypeptide 2; leukotriene B4 omega- hydroxylase;leukotriene-B4 20-monooxy- genase H24-147 TTGCAGGA 146 G protein- GPR153NM_207370 790 NP_997253 1047 GGACAAGA coupled TGC receptor 153 H24-148GATTGTGG 147 chloride CLIC6 XM_092804 335 XP_092804 377 CCAAGAAGintracellular NM_053277 336 NP_444507 378 TAC channel 6 H24-149 CCTCATTA148 G protein- GPR150 XM_094471 360 XP_094471 402 TCACCATG coupledNM_199243 361 NP_954713 403 CTC receptor 150 H24-150 CTGGTTAT 149FLJ16008 FLJ16008 NM_001001665 791 NP_001001665 1048 TGGCGGGT proteinATC H24-151 TTTGTGCT 150 G protein- GPR97 NM_170776 792 NP_740746 1049TCACCAAG coupled TGC receptor 97 H24-152 CTGCACAG 151 similar to N-LOC256135 XM_172523 793 XP_172523 1050 TCAACACG formyl peptide GTCreceptor H24-153 GCAGAGCC 152 LOC254502 LOC254502 XM_174355 794XP_174355 1051 AAATATCA GCC H24-154 GTTCAAGA 153 opsin 1 (cone OPN1MW,NM_000513 795 NP_000504 1052 AGCTGCGC pigments), OPN1LW NM_020061 796NP_064445 1053 CAC medium-wave- sensitive (color blindness, deutan) -opsin 1 (cone pigments), long-wave- sensitive (color blindness, protan)H24-155 CGTCTTCA 154 G protein- GPR30 NM_001505 797 NP_001496 1054TCAGCGTG coupled CAC receptor 30 H24-156 GCAGTTCC 155 opsin 1 (coneOPN1SW NM_001708 798 NP_001699 1055 AAGCTTGC pigments), ATC short-wave-sensitive (color blindness, tritan) H24-157 GTACCTGC 156 chemokine CCR3NM_178329 354 NP_847899 396 GCCACTTC (C—C motif) NM_001837 355 NP_001828397 TTC receptor 3 H24-158 GACCATCA 157 frizzled FZD7 NM_003507 799NP_003498 1056 CTATCCTG homolog 7 GCC (Drosophila) H24-159 GTCCTTCT 158G protein- GPR23 NM_005296 337 NP_005287 379 ACATCAAT coupled GCCreceptor 23 H24-160 GAAGAAGC 159 G protein- GPR64 NM_005756 338NP_005747 380 AACTGGGA coupled GCC receptor 64 H24-162 GTTCCAGA 160chemokine CCR9 NM_006641 800 NP_006632 1057 CCTTCATG (C—C motif)NM_031200 801 NP_112477 1058 TGC receptor 9 H24-163; CTGGACCG 161adrenomedullin ADMR NM_007264 802 NP_009195 1059 H24-226 AGCTGCTTreceptor GAC H24-165 GCAGAGCA 162 G protein- G2A NM_013345 803 NP_0374771060 TGGGCTTA coupled AGC receptor H24-167; AGCAGGCG 163 calcium CACNB3NM_000725 804 NP_000716 1061 H24-228 GAACATGT channel, TCC voltage-dependent, beta 3 subunit H24-169 CAACCTGT 164 galanin GALR2 NM_003857371 NP_003848 413 TCATCCTT receptor 2 AAC H24-170 ATTTGGTG 165chromosome C10orf112 XM_295865 805 XP_295865 1062 ACACGGCT 10 open GACreading frame 112 H24-171 CATATGGC 166 similar to LOC121456 XM_062645806 XP_062645 1063 TCTTCAAG solute carrier TAC family 9 (sodium/hydrogenexchanger), isoform 7; nonselective sodium potassium/pro- ton exchangerH24-172 TGGCACAG 167 acetyl- ACAT1 NM_000019 807 NP_000010 1064 TAACAGCTCoenzyme A GCC acetyltrans- ferase 1 (acetoacetyl (Coenzyme A thiolase)H24-173 GTTCTCTC 168 placental PGF NM_002632 808 NP_002623 1065 AGCACGTTgrowth factor, CGC vascular endothelial growth factor- related proteinH24-174 TGCAGACT 169 Rhesus blood RHAG NM_000324 809 NP_000315 1066TCAGTGCA group- GCC associated glycoprotein H24-175 CTTTCTGG 170 solutecarrier SLC39A1 NM_014437 810 NP_055252 1067 AAATCCTG family 39 (zincCCC transporter), member 3 H24-176 CAACACAG 171 UDP-N-acetyl- GALGTNM_001478 811 NP_001469 1068 CAGACACA alpha-D- GTC galactosamine:N-acetylneur- aminyl)-galac- tosylglucosyl- ceramide N- acetylgalactos-aminyltransfer- ase (GalNAc-T) H24-177 TGGGACCA 172 glycosylphos- GPLD1NM_001503 812 NP_001494 1069 GTGACTGC phatidylinositol AGC specificphospholipase D1 H24-178 AGCCAGAG 173 HMT1 hnRNP HRMT1L1 NM_001535 813NP_001526 1070 GAGTTTGT methyltransfer- NM_206962 814 NP_996845 1071 GGCase-like 1 (S. cerevisiae) H24-179 CTCGCAGG 174 GS3955 GS3955 NM_021643815 NP_067675 1072 AAATTCTG protein GAC H24-180 ATGCAGGT 175 solutecarrier SLC4A10 NM_022058 816 NP_071341 1073 CAGGTTGT family 4, TTCsodium bicarbonate transporter- like, member 10 H24-181 ATCAGGCC 176phosphoprotein C8FW NM_025195 817 NP_079471 1074 TTACATCC regulated byAGC mitogenic pathways H24-182 CTGTGGGA 177 similar to LOC165927XM_093541 818 XP_093541 1075 GAAACAGA hypothetical GAC protein,MNCb-4779 H24-183 CTCTGCGA 178 UDP- B3GALT6 NM_080605 819 NP_542172 1076CTACTACC Gal:betaGal TGC beta 1,3- galactosyltrans- ferase polypeptide 6H24-184 CTGATGAA 179 similar to LOC123326 XM_063593 820 XP_063593 1077GGCCTTCG NADH- ACC ubiquinone oxidoreductase PDSW subunit (Complex I-PDSW) (CI- PDSW) H24-185 ACCGTGGA 180 cytochrome CYP24 NM_000782 370NP_000773 412 GGCCTAT P450, CGC subfamily XXIV (vitamin D 24-hydroxylase) H24-186 TTCCAGCT 181 hypothetical FLJ11149 NM_018339 821NP_060809 1078 GATATATC protein CAC FLJ11149 H24-187 GCCAGACA 182 TTBK2SK453 822 SK453's protein 1131 CTGACAAG NM_173500 823 NP_775771 1079 TTCH24-188 TCGGCAGG 183 macrophage MST1 NM_020998 824 NP_066278 1080GCCAGCAT stimulating 1 TTC (hepatocyte growth factor- like) H24-189GTCTTCGA 184 histidine-rich HRG NM_000412 825 NP_000403 1081 CCCTCAGGglycoprotein AAC H24-190 TCAGAAGG 185 KIAA0943 APG4B NM_013325 826NP_037457 1082 TTGTGCAG protein NM_178326 827 NP_847896 1083 GAC H24-191CAACTTGC 186 amyloid beta APP NM_201414 828 NP_958817 1084 ATGACTAC (A4)precursor NM_000484 829 NP_000475 1085 GGC protein NM_201413 830NP_958816 1086 (protease nexin-II, Alzheimer disease) H24-192 TGTGCAAG187 protein PPP2CB NM_004156 831 NP_004147 1087 AGGTTCGT phosphatase 2TGC (formerly 2A), catalytic subunit, beta isoform H24-193 ACCAGTGG 188dual specificity DUSP5 NM_004419 358 NP_004410 400 TAAATGTC phosphatase5 AGC H24-194 CTCTGTAT 189 mitogen- MAP3K9 NM_033141 346 NP_149132 388CCCATTCC activated XM_027237 347 XP_027237 389 CTC protein kinase kinasekinase 9 H24-195 CTTCTTGA 190 mitogen- MAP3K1 XM_042066 832 XP_0420661088 AGCATCTA activated TGC protein kinase kinase kinase 1 H24-196CATGCTGT 191 leucyl/cystinyl LNPEP NM_005575 833 NP_005566 1089 TGTCCTTTaminopeptidase ACC H24-197 CGAAGATG 192 ribonuclease L RNASEL NM_021133834 NP_066956 1090 TTGACCTG (2′,5′- GTC oligoisoadenyl- ate synthetase-dependent) H24-198 TGTCCTGT 193 protein kinase, PRKAA2 NM_006252 835NP_006243 1091 TGGATGCA AMP- CAC activated, alpha 2 catalytic subunitH24-199 ATGCAGAC 194 tumor necrosis TNFRSF11A NM_003839 836 NP_0038301092 CCTGGACC factor receptor AAC superfamily, member 11a, activator ofNFKB H24-200 GTAGCACT 195 solute carrier SLC39A4 NM_017767 837 NP_0602371093 CTGCGACA family 39 (zinc NM_130849 838 NP_570901 1094 TGCtransporter), member 4 H24-201 CTGGAATG 196 succinate SDHC NM_003001 839NP_002992 1095 GGATCCGA dehydrogenase CAC complex, subunit C, integralmembrane protein, 15kD H24-202 GTTATTCT 197 nicotinamide NNMT NM_006169840 NP_006160 1096 TCCACCAT N-methyl- GGC transferase H24-203 TGCCAGCA198 ciliary CNTFR NM_001842 841 NP_001833 1097 GTCTCTTG neurotrophic ATCfactor receptor H24-204 GATCTTCC 199 insulin-like IGFBP5 NM_000599 842NP_000590 1098 GGCCCAAA growth factor CAC binding protein 5 H24-205AGCATGAC 200 UDP-glucose UGCGL2 NM_020121 843 NP_064506 1099 AGGAAACCceramide TGC glucosyltrans- ferase-like 2 H24-206 TCTTCTCT 201 similarto LOC136234 XM_069782 844 XP_069782 1100 GTGAACAT NADH ACCdehydrogenase subunit 1 H24-207 CCTTGTTG 202 similar to AADAC NM_207365845 NP_997248 1101 GCCAATGA Arylacetamide TTC deacetylase H24-208CCAGGTTT 203 azurocidin 1 AZU1 NM_001700 846 NP_001691 1102 GTCAACGT(cationic GAC antimicrobial protein 37) H24-209 TTCCAGCT 204 similar toLOC161785 XM_091124 847 XP_091124 1103 CAGTGCTA microtubule- ATCassociated proteins 1A/1B light chain 3 H24-210 AGCAGGAA 205 similar toLOC219756 XM_166676 848 XP_166676 1104 CATCTCTT MNK1 CGC H24-211CAAGTTCT 206 ATPase, ATP4B NM_000705 849 NP_000696 1105 CCTGCAAG H+/K+TTC exchanging, beta polypeptide H24-212 GAGCATGA 207 gamma- GABRR2NM_002043 850 NP_002034 1106 CCTTCGAT aminobutyric GGC acid (GABA)receptor, rho 2 H24-213 CTGGCAGA 208 a disintegrin- ADAMTS1 NM_006988851 NP_008919 1107 AGCAGCAC like and AAC metalloprotease (reprolysintype) with thrombospondin type 1 motif, 1 H24-214 TACCTGCT 209fibroblast FGF4 NM_002007 852 NP_001998 1108 GGGCATCA growth factor 4AGC (heparin secretory transforming protein 1, Kaposi sarcoma oncogene)H24-215 CTGGAAGT 210 interferon, IFNA8 NM_002170 853 NP_002161 1109CCTGTGTG alpha 8 ATC H24-216 GGACACCT 211 interleukin 19 IL19 NM_013371854 NP_037503 1110 TCCCAAAT NM_153758 855 NP_715639 1111 GTC H24-217GGCAGAAA 212 solute carrier SLC10A2 NM_000452 856 NP_000443 1112TTCCAGAG family 10 AGC (sodium/bile acid cotransporter family), member 2H24-218 CAACATCC 213 glutathione GSR NM_000637 857 NP_000628 1113CAACTGTG reductase GTC H24-219 TATCCTGA 214 potassium KCNG1 NM_002237372 NP_002228 414 CCTTCCTG voltage-gated NM_172318 373 NP_758529 415 CGCchannel, subfamily G, member 1 H24-220 TGTTTACC 215 fibroblast FGF21NM_019113 858 NP_061986 1114 AGTCCGAA growth factor GCC 21 H24-221AACTGTAC 216 similar to LOC131961 XM_067688 859 XP_067688 1115 CGCAGAGTINOSINE-5- TCC MONOPHOSPHATE DEHYDROGENASE 1 (IMP DEHYDRO- GENASE 1)(IMPDH-I) (IMPD 1) H24-222 AGCATGAT 217 topoisomerase TOP2B NM_001068860 NP_001059 1116 GATAGTTC (DNA) II beta CTC (180kD) H24-223 CAAGTGCC218 SFRS protein SRPK1 NM_003137 861 NP_003128 1117 GTATCATC kinase 1SK358 862 SK358's protein 1132 CAC H24-224 TATTCGTG 219 chromosome 9SgK071, SK521 863 SK521's protein 1133 CGGAGGAA open reading C9orf96NM_153710 864 NP_714921 1118 GAC frame 96 H24-227 GTACCTGC 220procollagen- P4HB NM_000918 865 NP_000909 1119 TGGTGGAG proline, 2- TTCoxoglutarate 4- dioxygenase (proline 4- hydroxylase), beta polypeptide(protein disulfide isomerase; thyroid hormone binding protein p55)

TABLE 1A SEQ TARGET Genbank KD TARGET Sequence ID Gene DNA/RNA (5′→3′)NO. TARGET Gene Name Symbol Accession CGTTGGCAGAATCATTTAC 247 Gprotein-coupled receptor 38 GPR38 NM_001507 TTCGCGGATGATGTACTTC 248 Gprotein-coupled receptor 38 GPR38 NM_001507 CTCTCAGTACTTTAACATC 249 Gprotein-coupled receptor 38 GPR38 NM_001507 CATAACAAAGGCATCGCCC 250 MAPkinase-interacting serine/threonine MKNK2, NM_199054 kinase 2 MNK2NM_017572 SK236 TTAATGATAGCCATCCAGC 251 casein kinase 1, gamma 1CSNK1G1, NM_022048 CK1g1 SK647 ATTCCAGGCCATTTATGGC 252 granzyme K(serine protease, granzyme GZMK NM_002104 3; tryptase II)TGTTAGACTACGTGATGAC 253 hypothetical protein FLJ14906 FLJ14906 NM_032859CAACCTCACCTACAGGATC 254 matrix metalloproteinase 23A - matrix MMP23A,NM_006983 metalloproteinase 23B MMP23B NM_004659 TCTACCCGATCAACCACAC 255matrix metalloproteinase 23A - matrix MMP23A, NM_006983metalloproteinase 23B MMP23B NM_004659 CCCTTGGAGGAACTATGCC 256 LOC254378LOC254378 XM_174812 CAACTCAGACAACTGCATC 257 LOC254378 LOC254378XM_174812 TCATTGTAAGGCTGTGGCC 258 similar to bile salt-dependent lipaseLOC138529 XM_070951 oncofetal isoform GGACCAATGGGAGATAGAC 259fyn-related kinase FRK NM_002031 AGCTTATCCAGCTTTATGC 260 fyn-relatedkinase FRK NM_002031 GTGCATTAACAAGAAGAAC 261 unc-51-like kinase 1 (C.elegans) ULK1 NM_003565 ACCTGCAGCCTTCACTTTC 262 chemokine (C—C motif)receptor 1 CCR1 NM_001295 TCCCTTCTGGATCGACTAC 263 chemokine (C—C motif)receptor 1 CCR1 NM_001295 CAATTATGAGTCCACGGTC 264 olfactory receptor,family 1, subfamily OR1A2 NM_012352 A, member 2 NM_014565AACGATGGGCATGTATTTC 265 olfactory receptor, family 1, subfamily OR1A2NM_012352 A, member 2 TGTCTCCTATGTTCAGGTC 266 olfactory receptor, family1, subfamily OR1A2 NM_012352 A, member 2 GTATGGACAGAACTGGCTC 267sentrin/SUMO-specific protease 3 SENP3 NM_015670 GATGAACATGTATGGAGAC 268sentrin/SUMO-specific protease 3 SENP3 NM_015670 ATTCCTTCAAACGTATGGC 269sentrin/SUMO-specific protease 3 SENP3 NM_015670 GCTCCTGGAGAACATGTAC 270taste receptor, type 1, member 3 TAS1R3 XM_371210 ACCAACCTCAGAGGTTCTC271 thousand and one amino acid protein TAO1 NM_016151 kinase - prostatederived STE20-like NM_004783 kinase PSK AAGCGGACCTACAAACTTC 272 thousandand one amino acid protein TAO1 NM_016151 kinase - prostate derivedSTE20-like NM_004783 kinase PSK CGTCTACATGTACGTGTTC 273 solute carrierfamily 16 (monocar- SLC16A3 NM_004207 boxylic acid transporters), member3 GTGTGTACATCAACTGTTC 274 androgen-regulated short-chain ARSDR1NM_016026 dehydrogenase/reductase 1 GGCACAGTCCAATCTGAAC 275androgen-regulated short-chain ARSDR1 NM_016026 dehydrogenase/reductase1 CGCAAGTTCTACTCCATCC 276 RAS, dexamethasone-induced 1 RASD1 NM_016084GGTGTTCAGTCTGGACAAC 277 RAS, dexamethasone-induced 1 RASD1 NM_016084GTGTTCAGTCTGGACAACC 278 RAS, dexamethasone-induced 1 RASD1 NM_016084ACTTGAACTCTCTCCACAC 279 chromosome 20 open reading frame 121 C20orf121NM_024331 GTCTTCAATAACTTGAAGC 280 chromosome 20 open reading frame 121C20orf121 NM_024331 CCACAACAAGCACGTGTTC 281 HMT1 hnRNPmethyltransferase-like 3 HRMT1L3 NM_019854 (S. cerevisiae)ATCAATCGAAAGATTCTTC 282 interleukin-1 receptor-associated IRAK2NM_001570 kinase 2 SK180 CGACGTTGACAATTCCAGC 283 interleukin-1receptor-associated IRAK2 NM_001570 kinase 2 SK180 GCAGAGTTGCAGATTTGTC284 interleukin-1 receptor-associated IRAK2 NM_001570 kinase 2 SK180TCAGCATTCCTACGATAAC 285 glutamate receptor, ionotropic, N- GRIN2ANM_000833 methyl D-aspartate 2A CCGGCAGAAGGATAACCTC 286 glutamatereceptor, ionotropic, N- GRIN2A NM_000833 methyl D-aspartate 2ACCAGAACTGTGAAGTTTAC 287 glutamate receptor, ionotropic, N- GRIN2ANM_000833 methyl D-aspartate 2A GCATGGCAAGAAAGTTAAC 288 glutamatereceptor, ionotropic, N- GRIN2A NM_000833 methyl D-aspartate 2AAGCATGTTATGCCTTATGC 289 glutamate receptor, ionotropic, N- GRIN2ANM_000833 methyl D-aspartate 2A TCATTGTTTCTGCCATAGC 290 glutamatereceptor, ionotropic, N- GRIN2A NM_000833 methyl D-aspartate 2AGATCTTTAACCAGCCTGAC 291 hypothetical protein FLJ22955 FLJ22955 NM_024819GCACACCGTGGTAGTATAC 292 hypothetical protein FLJ22955 FLJ22955 NM_024819GTCCTTCCACATCAAGAAC 293 tumor endothelial marker 5 precursor TEM5NM_032777 TGCAACCTCTTACCCAAGC 294 chloride intracellular channel 6 CLIC6NM_053277 CTCAATCCCTTCGTCTACC 295 G protein-coupled receptor 150 GPR150NM_199243 CGTGGTCTACGCGTTCTAC 296 LOC167417 XM_094471 XM_094471CATTATCACCATGCTCGGC 297 G protein-coupled receptor 150 GPR150 NM_199243TCTCTCTTCCTATCAATCC 298 chemokine (C—C motif) receptor 3 CCR3 NM_178329NM_001837 CCAAACGTGTCTGGAAGAC 299 G protein-coupled receptor 23 GPR23NM_005296 GTACCTGTAGCCATCTAAC 300 G protein-coupled receptor 64 GPR64NM_005756 GCTAGTGAATAATGATTGC 301 G protein-coupled receptor 64 GPR64NM_005756 ACACGACTATAAGTCTAAC 302 G protein-coupled receptor 64 GPR64NM_005756 ACATTGCAATGGACAACAC 303 KIAA0943 protein APG4B NM_013325NM_178326 GGAATATCCTGAGTGTTGC 304 dual specificity phosphatase 5 DUSP5NM_004419 TGCCAGAGGGATGAACTAC 305 mitogen-activated protein kinasekinase MAP3K9 NM_033141 kinase 9 ATGGAAGACTGCTGGAATC 306mitogen-activated protein kinase kinase MAP3K9 NM_033141 kinase 9GAGCGCTTCAAACGAGATC 307 mitogen-activated protein kinase kinase MAP3K9NM_033141 kinase 9 ACCTGTCCCTAGATTCTTC 308 KIAA0943 protein APG4BNM_013325 ACGCATCTTGGCAAAGAGC 309 casein kinase 1, gamma 1 CSNK1G1-NM_022048 CK1g1 SK647 ATTCCAGGGTTTATGTGTC 310 peptidylprolyl isomerase APPIA, XM_372328 LOC390006, XM_371409 LOC388817, NM_203431 LOC442744,XM_499491 LOC342541, XM_292596 LOC344178, XM_292963 LOC388687, XM_371304LOC391352, XM_372916 LOC256374, XM_170597 LOC388686, XM_371302LOC122335, NM_021130 LOC390827, XM_063084 LOC390956, XM_497571LOC442362, XM_372741 LOC343384, NM_203430 COAS2, XM_498254 LOC44006,XM_291544 LOC392352 NM_178230 XM_495896 XM_373301 XM_060887 XM_377444CAACAGTGCATCTCTTATC 311 liver-specific organic anion trans- LST-3NM_001009562 porter 3 (XM_292093) CAGCATCTACCTCCTGAAC 312 chemokine (C—Cmotif) receptor 1 CCR1 NM_001295 CAGCGCTCTCAATCCCTTC 313 Gprotein-coupled receptor 150 GPR150 NM_199243 CATCGACTATATAGCAGGC 314carboxyl ester lipase (bile salt- CEL NM_001807 stimulated lipase)CCAAGAGTCTATTCAAAGC 315 olfactory receptor, family 1, subfamily OR1A2NM_012352 A, member 2 CCACTAATGTCAACAATGC 316 G protein-coupled receptor23 GPR23 NM_005296 CCACTCGTCAGATGTTTGC 317 LOC254378 LOC254378 XM_174812CCCTGTCATTGATGGAGAC 318 carboxyl ester lipase (bile salt- CEL NM_001807stimulated lipase) CCGAGCCATATACTTGACC 319 chromosome 20 open readingframe 121 C20orf121 NM_024331 CCTAGAGCTGATTGAGTTC 320 MAPkinase-interacting serine/threonine MKNK2- NM_199054 kinase 2 MNK2NM_017572 SK236 CCTCGACACCAAGTCTTGC 321 RAS, dexamethasone-induced 1RASD1 NM_016084 CGCTCTTTAACATGAATGC 322 thousand and one amino acidprotein TAO1 NM_016151 kinase - prostate derived STE20-like NM_004783kinase PSK CGTGGACATGGAGTACGAC 323 taste receptor, type 1, member 3TAS1R3 XM_371210 CTCGTAATGAGACTATAGC 324 androgen-regulated short-chainARSDR1 NM_016026 dehydrogenase/reductase 1 CTGCAAATCTTCAGGTTTC 325 Gprotein-coupled receptor 64 GPR64 NM_005756 GAAGCACGATTTGGAGGTC 326unc-51-like kinase 1 (C. elegans) ULK1 NM_003565 GATGATGAAGGAGACGTTC 327hypothetical protein FLJ22955 FLJ22955 NM_024819 GATTTGGTTATAAGGGTTC 328peptidylprolyl isomerase A LOC126170 XM_497621 LOC388817 XM_371409 PPIANM_203431 KBTBD9 XM_496546 LOC256374 XM_170597 LOC131055 XM_067176LOC390956 NM_021130 LOC391062 XM_372741 LOC343384 XM_372785 LOC401859NM_203430 XM_291544 XM_377444 GCTACTGCCCTATATGATC 329 solute carrierfamily 39 (zinc SLC39A4 NM_017767 transporter), member 4 NM_130849TCCGGTTCTATTTGATCGC 330 tumor endothelial marker 5 precursor TEM5NM_032777 TCGCCCTTCCTATTCCTTC 331 mitogen-activated protein kinasekinase MAP3K9 NM_033141 kinase 9 TGTACGTGTTCATCCTGGC 332 solute carrierfamily 16 (monocar- SLC16A3 NM_004207 boxylic acid transporters), member3 TGTGACATTATGCCTTTGC 333 olfactory receptor, family 1, subfamily OR1A2NM_102352 A, member 2

TABLE 2 KD TARGET TARGET Genbank DNA/RNA Genbank TARGET SEQ ID GeneDNA/RNA SEQ ID Polypeptide Polypeptide Id NO. Symbol Class Accession NO.Accession SEQ ID NO. H24-148 147 CLIC6 Ion Channel XM_092804 335XP_092804 377 NM_053277 336 NP_444507 378 H24-159 158 GPR23 GPCRNM_005296 337 NP_005287 379 H24-160 159 GPR64 GPCR NM_005756 338NP_005747 380 H24-142 141 GRIN2A Receptor NM_000833 339 NP_000824 381H24-024 26 MMP23A Protease NM_004659 340 NP_004650 382 MMP23B NM_006983341 NP_008914 383 H24-054 54 CCR1 GPCR NM_001295 342 NP_001286 384H24-128 127 HRMT1L3 Enzyme NM_019854 343 NP_062828 385 H24-138 137 IRAK2Kinase NM_001570 344 NP_001561 386 SK180 345 SK180's protein 387 H24-194189 MAP3K9 Kinase NM_033141 346 NP_149132 388 XM_027237 347 XP_027237389 H24-004 6 MKNK2 Kinase SK236 348 SK236's protein 390 NM_017572 349NP_060042 391 NM_199054 350 NP_951009 392 H24-097 96 TAOK2, KinaseNM_016151 351 NP_057235 393 PSK, NM_004783 352 NP_004774 394 TAO1H24-021 23 C13orf6, Protease NM_032859 353 NP_116248 395 FLJ14906H24-157 156 CCR3 GPCR NM_178329 354 NP_847899 396 NM_001837 355NP_001828 397 H24-006 8 CSNK1G1 Kinase NM_001011664 356 NP_001011664 398(SK647) NM_022048 357 NP_071331 399 H24-193 188 DUSP5 PhosphataseNM_004419 358 NP_004410 400 H24-143 142 FLJ22955 Kinase NM_024819 359NP_079095 401 H24-149 148 GPR150 GPCR XM_094471 360 XP_094471 402NM_199243 361 NP_954713 403 H24-014 16 GZMK Protease NM_002104 362NP_002095 404 H24-064; 63 OR1A1 GPCR NM_014565 363 NP_055380 405 H24-164H24-064; 63 OR1A2 GPCR NM_012352 364 NP_036484 406 H24-164 H24-112 111RASD1 Enzyme NM_016084 365 NP_057168 407 H24-099 98 SLC16A3 TransporterNM_004207 366 NP_004198 408 H24-049 1 AVPR1B GPCR NM_000707 367NP_000698 409 H24-007 9 caMLCK Kinase SK536 368 SK536's protein 410NM_182493 369 NP_872299 411 (AJ247087) H24-185 180 CYP24A1 CytochromeNM_000782 370 NP_000773 412 P450 H24-169 164 GALR2 GPCR NM_003857 371NP_003848 413 H24-219 214 KCNG1 Ion Channel NM_002237 372 NP_002228 414NM_172318 373 NP_758529 415 H24-094 93 PAK6 Kinase NM_020168 374NP_064553 416 SK429 375 SK429's protein 417 H24-041 42 PIK3C2B KinaseNM_002646 376 NP_002637 418 H24-008 10 ROCK2 Kinase NM_004850 600NP_004841 601

TABLE 2A Seq ID protein Accession Name Protein Segment segment NP_444507CLIC6 O—ClC: intracellular 419 chloride channel protein NP_005287 GPR23Extracellular domain 420 Transmembrane domain 421 Intracellular domain422 Transmembrane domain 423 Extracellular domain 424 Transmembranedomain 425 Intracellular domain 426 Transmembrane domain 427Extracellular domain 428 Transmembrane domain 429 Intracellular domain430 Transmembrane domain 431 Extracellular domain 432 Transmembranedomain 433 Intracellular domain 434 NP_005747 GPR64 Extracellular domain435 Transmembrane domain 436 Intracellular domain 437 Transmembranedomain 438 Extracellular domain 439 Transmembrane domain 440Intracellular domain 441 Transmembrane domain 442 Extracellular domain443 Transmembrane domain 444 Intracellular domain 445 Transmembranedomain 446 Extracellular domain 447 Transmembrane domain 448Intracellular domain 449 NP_000824 GRIN2A Lig_chan 450 NP_004650 MMP23AZnMc 451 Peptidase_M10 452 ShKToxin domain 453 Immunoglobulin 454NP_008914 MMP23B ZnMc 455 Peptidase_M10 456 ShKToxin domain 457Immunoglobulin 458 NP_001286 CCR1 Extracellular domain 459 Transmembranedomain 460 Intracellular domain 461 Transmembrane domain 462Extracellular domain 463 Transmembrane domain 464 Intracellular domain465 Transmembrane domain 466 Extracellular domain 467 Transmembranedomain 468 Intracellular domain 469 Transmembrane domain 470Extracellular domain 471 Transmembrane domain 472 Intracellular domain473 NP_062828 HRMT1L3 Predicted RNA methylase 474 NP_001561 IRAK2 Deathdomain 475 SK180's protein protein kinase 476 NP_149132 MAP3K9 SH3_1 477XP_027237 protein kinase 478 SK236's protein MKNK2 protein kinase 479NP_060042 MKNK2 protein kinase 480 NP_951009 NP_057235 TAOK2 = proteinkinase 481 NP_004774 PSK = TAO1 NP_116248 C13orf6 = Alpha/beta hydrolase482 FLJ14906 fold-1 NP_847899 CCR3 Rhodopsin-like GPCR 483 NP_001828superfamily Extracellular domain 484 Transmembrane domain 485Intracellular domain 486 Transmembrane domain 487 Extracellular domain488 Transmembrane domain 489 Intracellular domain 490 Transmembranedomain 491 Extracellular domain 492 Transmembrane domain 493Intracellular domain 494 Transmembrane domain 495 Extracellular domain496 Transmembrane domain 497 Intracellular domain 498 NP_001011664CSNK1G1 protein kinase 499 NP_071331 NP_004410 DUSP5 Rhodanese-like 500Dual specificity protein 501 phosphatase NP_079095 FLJ22955Dephospho-CoA kinase 502 XP_094471 GPR150 Intracellular domain 503Transmembrane domain 504 Extracellular domain 505 Transmembrane domain506 Intracellular domain 507 Transmembrane domain 508 Extracellulardomain 509 Transmembrane domain 510 Intracellular domain 511 NP_954713GPR150 Extracellular domain 512 Transmembrane domain 513 Intracellulardomain 514 Transmembrane domain 515 Extracellular domain 516Transmembrane domain 517 Intracellular domain 518 Transmembrane domain519 Extracellular domain 520 Transmembrane domain 521 Intracellulardomain 522 NP_002095 GZMK Peptidase S1 and S6, 523 chymotrypsin/HapNP_055380 OR1A1 Extracellular domain 524 Transmembrane domain 525Intracellular domain 526 Transmembrane domain 527 Extracellular domain528 Transmembrane domain 529 Intracellular domain 530 Transmembranedomain 531 Extracellular domain 532 Transmembrane domain 533Intracellular domain 534 Transmembrane domain 535 Extracellular domain536 Transmembrane domain 537 Intracellular domain 538 NP_036484 OR1A2Extracellular domain 539 Transmembrane domain 540 Intracellular domain541 Transmembrane domain 542 Extracellular domain 543 Transmembranedomain 544 Intracellular domain 545 Transmembrane domain 546Extracellular domain 547 Transmembrane domain 548 Intracellular domain549 Transmembrane domain 550 Extracellular domain 551 Transmembranedomain 552 Intracellular domain 553 NP_057168 RASD1 Ras small GTPase,554 Ras type NP_004198 SLC16A3 Major facilitator 555 superfamily MFS_1NP_000698 AVPR1B Extracellular domain 556 Transmembrane domain 557Intracellular domain 558 Transmembrane domain 559 Extracellular domain560 Transmembrane domain 561 Intracellular domain 562 Transmembranedomain 563 Extracellular domain 564 Transmembrane domain 565Intracellular domain 566 Transmembrane domain 567 Extracellular domain568 Transmembrane domain 569 Intracellular domain 570 SK536's proteincaMLCK Protein kinase 571 NP_872299 caMLCK Serine/threonine 572 proteinkinase NP_000773 CYP24A1 Cytochrome P450 573 NP_003848 GALR2Extracellular domain 574 Transmembrane domain 575 Intracellular domain576 Transmembrane domain 577 Extracellular domain 578 Transmembranedomain 579 Intracellular domain 580 Transmembrane domain 581Extracellular domain 582 Transmembrane domain 583 Intracellular domain584 Transmembrane domain 585 Extracellular domain 586 Transmembranedomain 587 Intracellular domain 588 NP_002228 KCNG1 K+ channel 589tetramerisation Ion transport 590 NP_758529 KCNG1 K+ channel 591tetramerisation NP_064553 PAK6 PAK-box/P21-Rho-binding 592 SK429'sprotein Serine/threonine 593 protein kinase NP_002637 PIK3C2BPhosphoinositide 594 3-kinase, ras-binding Phosphoinositide 5953-kinase, C2 Phosphoinositide 596 3-kinase accessory region PIKPhosphatidylinositol 597 3- and 4-kinase, catalytic Phox-like 598 C2 599NP_004841 ROCK2 Serine/threonine 602 protein kinase Protein kinase,C-terminal 603 PKN/rhophilin/rhotekin 604 rho-binding repeatPleckstrin-like 605 Protein kinase C, 606 phorbol ester/diacyl- glycerolbinding

Table 3 lists the preferred loop sequence for a shRNA.

TABLE 3 Name Sequence SEQ ID NO: Loop region GTTTGCTATAAC 334

The present invention relates to a method for assaying for compoundsthat induce osteoblast differentiation, comprising contacting thecompound with a polypeptide comprising an amino acid sequence of thepolypeptides of SEQ ID NO: 377-599, 601-606, 867-1119, 1123-1133(“TARGETS”) under conditions that allow said polypeptide to bind to thecompound, and detecting the formation of a complex between thepolypeptide and the compound. One preferred means of measuring thecomplex formation is to determine the binding affinity of said compoundto said polypeptide.

More particularly, the invention relates to a method for identifying anagent that induces differentiation of undifferentiated mammalian cellsinto osteoblasts, the method comprising further:

-   -   (a) contacting a population of undifferentiated vertebrate cells        with one or more of said compound that exhibits binding affinity        for said TARGETS, and    -   (b) measuring a compound-polypeptide property related to the        differentiation of said cells into osteoblasts.

The compound-polypeptide property referred to above is related to thedifferentiation of cells into osteoblasts, and is a measurablephenomenon chosen by the person of ordinary skill in the art. Themeasurable property may e.g. be the binding affinity for a peptidedomain of the polypeptide TARGET or the level of any one of a number ofbiochemical marker levels of osteoblast differentiation. Osteoblastdifferentiation can e.g. be measured by measuring the level of enzymesthat are induced during the differentiation process, such as alkalinephosphatase, type-1 collagen, osteocalcin and osteopontin. The alkalinephosphatase activity can be measured by adding methylumbelliferylheptaphosphate (MUP) solution (Sigma) to the cells. The fluorescencegenerated upon cleavage of the MUP substrate by the AP activity ismeasured on a fluorescence plate reader (Fluostar, BMG).

In a preferred embodiment of the invention, the polypeptide TARGETcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 377-599, 601-606, 867-1119, 1123-1133 (Tables 1, 2, and 2A).In another preferred embodiment of the invention, the polypeptide TARGETcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 377-599, 601-606 (Tables 2 and 2A).

A preferred class of the polypeptides used as TARGETS are G-ProteinCoupled Receptors (GPCR), wherein the expression and/or activity of saidGPCR may be measured by determining the level of any one of the secondmessengers cyclic AMP, Ca²⁺ or both. Preferably, the level of the secondmessenger is determined with a reporter gene under the control of apromoter that is responsive to the second messenger. More preferably,the promoter is a cyclic AMP-responsive promoter, an NF-KB responsivepromoter, or a NF-AT responsive promoter. In another preferredembodiment, the reporter gene is selected from the group consisting of:alkaline phosphatase, GFP, eGFP, dGFP, luciferase and b-galactosidase.

Other preferred classes of polypeptides used as TARGETS include kinasesand phosphatases. Kinases are enzymes that transfer phosphate groupsfrom high-energy donor molecules, such as ATP, to specific targetmolecules (substrates); the process is termed phosphorylation.Phosphatases remove phosphate groups from substrates. The activity ofkinases or phosphatases may be measured by determining the level ofphosphorylation or dephosphorylation of a substrate of said polypeptide.It is well known in the art how to measure this activity by using therespective substrate and to perform assays in which the substrate getsphosphorylated or dephosphorylated upon activation of the kinase orphosphatase. This means that the activity of the kinase or phosphatasecan easily be scored through the phosphorylation status of itssubstrate.

Another preferred class of polypeptides used as TARGETS are proteases.Proteases are enzymes that break peptide bonds between amino acids ofproteins; the process is called proteolytic cleavage. Protease activitycan be measured through the level of cleavage of the respectivesubstrate, which is a preferred method according to the invention todetermine the activity level of the protease. Classically, substratesare used in which a fluorescent group is linked to a quencher through apeptide sequence that is a substrate that can be cleaved by the targetprotease. Cleavage of the linker separates the fluorescent group andquencher, giving rise to an increase in fluorescence.

Another preferred class of polypeptides used as TARGETS are ionchannels. Ion channels are membrane protein complexes and their functionis to facilitate the diffusion of ions across biological membranes.Membranes, or phospholipid bilayers, build a hydrophobic, low dielectricbarrier to hydrophilic and charged molecules. Ion channels provide ahigh conducting, hydrophilic pathway across the hydrophobic interior ofthe membrane. The activity of an ion channel can be measured usingclassical patch clamping. High-throughput fluorescence-based ortracer-based assays are also widely available to measure ion channelactivity. These fluorescent-based assays screen compounds on the basisof their ability to either open or close an ion channel thereby changingthe concentration of specific fluorescent dyes across a membrane. In thecase of the tracer based assay, the changes in concentration of thetracer within and outside the cell are measured by radioactivitymeasurement or gas absorption spectrometry.

Depending on the choice of the skilled artisan, the present assay methodmay be designed to function as a series of measurements, each of whichis designed to determine whether the drug candidate compound is indeedacting on the polypeptide to thereby induce the differentiation ofundifferentiated cells into osteoblasts. For example, an assay designedto determine the binding affinity of a compound to the polypeptide, orfragment thereof, may be necessary, but not sufficient, to ascertainwhether the test compound would be useful for increasing mean bonedensity when administered to a subject. Nonetheless, such bindinginformation would be useful in identifying a set of test compounds foruse in an assay that would measure a different property, further downthe biochemical pathway, such as bone mineralization, assayed bymeasuring the amount of deposited calcium. Such second assay may bedesigned to confirm that the test compound, having binding affinity forthe polypeptide, actually induces the differentiation ofundifferentiated cells into osteoblasts. Suitable controls should alwaysbe in place to insure against false positive readings.

The order of taking these measurements is not believed to be critical tothe practice of the present invention, which may be practiced in anyorder. For example, one may first perform a screening assay of a set ofcompounds for which no information is known respecting the compounds'binding affinity for the polypeptide. Alternatively, one may screen aset of compounds identified as having binding affinity for a polypeptidedomain, or a class of compounds identified as being an inhibitor of thepolypeptide. However, for the present assay to be meaningful to theultimate use of the drug candidate compounds, a measurement of bonealkaline phosphatase levels or bone mineralization is necessary.Validation studies including controls, and measurements of bindingaffinity to the polypeptides of the invention are nonetheless useful inidentifying a compound useful in any therapeutic or diagnosticapplication.

The binding affinity of the compound with the polypeptide TARGET can bemeasured by methods known in the art, such as using surface plasmonresonance biosensors (Biacore), by saturation binding analysis with alabeled compound (e.g. Scatchard and Lindmo analysis), by differentialUV spectrophotometer, fluorescence polarization assay, FluorometricImaging Plate Reader (FLIPR®) system, Fluorescence resonance energytransfer, and Bioluminescence resonance energy transfer. The bindingaffinity of compounds can also be expressed in dissociation constant(Kd) or as IC50 or EC50. The IC50 represents the concentration of acompound that is required for 50% inhibition of binding of anotherligand to the polypeptide. The EC50 represents the concentrationrequired for obtaining 50% of the maximum effect in any assay thatmeasures TARGET function. The dissociation constant, Kd, is a measure ofhow well a ligand binds to the polypeptide, it is equivalent to theligand concentration required to saturate exactly half of thebinding-sites on the polypeptide. Compounds with a high affinity bindinghave low Kd, IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM;a moderate to low affinity binding relates to a high Kd, IC50 and EC50values, i.e. in the micromolar range.

The present assay method may also be practiced in a cellular assay, Ahost cell expressing TARGET can be a cell with endogenous expression ora cell over-expressing the TARGET e.g. by transduction. When theendogenous expression of the polypeptide is not sufficient to determinea baseline that can easily be measured, one may use host cells thatover-express TARGET. Over-expression has the advantage that the level ofthe TARGET substrate end products is higher than the activity level byendogenous expression. Accordingly, measuring such levels usingpresently available techniques is easier. In such cellular assay, thebiological activity of TARGET may be measured by following theproduction of bone alkaline phosphatase (BAP) or bone mineralization.

The present invention further relates to a method for identifying acompound that induces differentiation of undifferentiated mammaliancells into osteoblasts, comprising:

-   -   (a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        377-599, 601-606, 867-1119, 1123-1133;    -   (b) determining the binding affinity of the compound to the        polypeptide;    -   (c) contacting a population of mammalian cells expressing said        polypeptide with the compound that exhibits a binding affinity        of at least 10 micromolar; and    -   (d) identifying the compound that induces the differentiation of        said undifferentiated cells.

For high-throughput purposes, libraries of compounds may be used such asantibody fragment libraries, peptide phage display libraries, peptidelibraries (e.g. LOPAP™, Sigma Aldrich), lipid libraries (BioMol),synthetic compound libraries (e.g. LOPAC™, Sigma Aldrich) or naturalcompound libraries (Specs, TimTec).

Preferred drug candidate compounds are low molecular weight compounds.Low molecular weight compounds, i.e. with a molecular weight of 500Dalton or less, are likely to have good absorption and permeation inbiological systems and are consequently more likely to be successfuldrug candidates than compounds with a molecular weight above 500 Dalton(Lipinski et al. (1997)). Peptides comprise another preferred class ofdrug candidate compounds. Peptides may be excellent drug candidates andthere are multiple examples of commercially valuable peptides such asfertility hormones and platelet aggregation inhibitors. Naturalcompounds are another preferred class of drug candidate compound. Suchcompounds are found in and extracted from natural sources, and which maythereafter be synthesized. The lipids are another preferred class ofdrug candidate compound.

Another preferred class of drug candidate compounds is an antibody. Thepresent invention also provides antibodies directed against a TARGET.These antibodies may be endogenously produced to bind to the TARGETwithin the cell, or added to the tissue to bind to TARGET polypeptidepresent outside the cell. These antibodies may be monoclonal antibodiesor polyclonal antibodies. The present invention includes chimeric,single chain, and humanized antibodies, as well as FAb fragments and theproducts of a FAb expression library, and Fv fragments and the productsof an Fv expression library.

In certain embodiments, polyclonal antibodies may be used in thepractice of the invention. The skilled artisan knows methods ofpreparing polyclonal antibodies. Polyclonal antibodies can be raised ina mammal, for example, by one or more injections of an immunizing agentand, if desired, an adjuvant. Typically, the immunizing agent and/oradjuvant will be injected in the mammal by multiple subcutaneous orintraperitoneal injections. Antibodies may also be generated against theintact TARGET protein or polypeptide, or against a fragment, derivativesincluding conjugates, or other epitope of the TARGET protein orpolypeptide, such as the TARGET embedded in a cellular membrane, or alibrary of antibody variable regions, such as a phage display library.

It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants that may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). One skilled in the art withoutundue experimentation may select the immunization protocol.

In some embodiments, the antibodies may be monoclonal antibodies.Monoclonal antibodies may be prepared using methods known in the art.The monoclonal antibodies of the present invention may be “humanized” toprevent the host from mounting an immune response to the antibodies. A“humanized antibody” is one in which the complementarity determiningregions (CDRs) and/or other portions of the light and/or heavy variabledomain framework are derived from a non-human immunoglobulin, but theremaining portions of the molecule are derived from one or more humanimmunoglobulins. Humanized antibodies also include antibodiescharacterized by a humanized heavy chain associated with a donor oracceptor unmodified light chain or a chimeric light chain, or viceversa. The humanization of antibodies may be accomplished by methodsknown in the art (see, e.g. Mark and Padlan, (1994) “Chapter 4.Humanization of Monoclonal Antibodies”, The Handbook of ExperimentalPharmacology Vol. 113, Springer-Verlag, New York). Transgenic animalsmay be used to express humanized antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter,(1991) J. Mol. Biol. 227:381-8; Marks et al. (1991). J. Mol. Biol.222:581-97). The techniques of Cole, et al. and Boerner, et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole, etal. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77; Boerner, et al (1991). J. Immunol., 147(1):86-95).

Techniques known in the art for the production of single chainantibodies can be adapted to produce single chain antibodies to theTARGET polypeptides and proteins of the present invention. Theantibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain cross-linking.Alternatively; the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcross-linking.

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens and preferably for a cell-surface protein or receptor orreceptor subunit. In the present case, one of the binding specificitiesis for one domain of the TARGET; the other one is for another domain ofthe same or different TARGET.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, (1983) Nature 305:537-9). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. Affinitychromatography steps usually accomplish the purification of the correctmolecule. Similar procedures are disclosed in Trauneeker, et al. (1991)EMBO J. 10:3655-9.

According to another preferred embodiment, the assay method uses a drugcandidate compound identified as having a binding affinity for a TARGET,and/or has already been identified as having down-regulating activitysuch as antagonist activity vis-à-vis one or more TARGET.

The present invention further relates to a method for inducingdifferentiation of undifferentiated mammalian cells into osteoblastscomprising contacting said cells with an expression inhibitory agentcomprising a polynucleotide sequence that complements at least about 17nucleotides of the polyribonucleotide comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO: 335-376, 600, 607-866,1120-1122.

Another aspect of the present invention relates to a method for inducingthe differentiation of undifferentiated mammalian cells intoosteoblasts, comprising by contacting said cell with anexpression-inhibiting agent that inhibits the translation in the cell ofa polyribonucleotide encoding a TARGET polypeptide. A particularembodiment relates to a composition comprising a polynucleotideincluding at least one antisense strand that functions to pair the agentwith the TARGET mRNA, and thereby down-regulate or block the expressionof TARGET polypeptide. The inhibitory agent preferably comprisesantisense polynucleotide, a ribozyme, and a small interfering RNA(siRNA), wherein said agent comprises a nucleic acid sequencecomplementary to, or engineered from, a naturally-occurringpolynucleotide sequence encoding a portion of a polypeptide comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:377-418, 601, 867-1119, 1123-1133. In a preferred embodiment theexpression-inhibiting agent is complementary to a polynucleotidesequence selected from the group consisting of SEQ ID NO: 335-376, 600,607-866, 1120-1122. In another preferred embodiment theexpression-inhibiting agent is complementary to a polynucleotidesequence selected from the group consisting of SEQ ID NO: 1-220,247-333.

An embodiment of the present invention relates to a method wherein theexpression-inhibiting agent is selected from the group consisting ofantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for SEQ ID NO: 377-418, 601,867-1119, 1123-1133, a small interfering RNA (siRNA, preferably shRNA,)that is sufficiently complementary to a portion of thepolyribonucleotide coding for SEQ ID NO: 377-418, 601, 867-1119,1123-1133, such that the siRNA, preferably shRNA, interferes with thetranslation of the TARGET polyribonucleotide to the TARGET polypeptide.Preferably the expression-inhibiting agent is an antisense RNA,ribozyme, antisense oligodeoxynucleotide, or siRNA, preferably shRNA,complementary to a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 335-376, 600, 607-866, 1120-1122.

A special embodiment of the present invention relates to a methodwherein the expression-inhibiting agent is a nucleic acid expressing theantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for SEQ ID NO: 377-418, 601, asmall interfering RNA (siRNA, preferably shRNA,) that is sufficientlycomplementary to a portion of the polyribonucleotide corresponding toSEQ ID NO: 377-418, 601, such that the siRNA, preferably shRNA,interferes with the translation of the TARGET polyribonucleotide to theTARGET polypeptide. Preferably the nucleotide sequence is complementaryto a polynucleotide selected from the group consisting of SEQ ID NO:335-376, 600. In another preferred embodiment nucleotide sequence iscomplementary to a polynucleotide selected from the group consisting ofSEQ ID NO: 1, 6, 8, 9, 10, 16, 23, 26, 42, 54, 63, 93, 96, 98, 111, 127,137, 141, 142, 147, 148, 156, 158, 159, 164, 180, 188, 189, 214.

The down regulation of gene expression using antisense nucleic acids canbe achieved at the translational or transcriptional level. Antisensenucleic acids of the invention are preferably nucleic acid fragmentscapable of specifically hybridizing with all or part of a nucleic acidencoding a TARGET polypeptide or the corresponding messenger RNA. Inaddition, antisense nucleic acids may be designed which decreaseexpression of the nucleic acid sequence capable of encoding a TARGETpolypeptide by inhibiting splicing of its primary transcript. Any lengthof antisense sequence is suitable for practice of the invention so longas it is capable of down-regulating or blocking expression of a nucleicacid coding for a TARGET. Preferably, the antisense sequence is at leastabout 17 nucleotides in length. The preparation and use of antisensenucleic acids, DNA encoding antisense RNAs and the use of oligo andgenetic antisense is known in the art.

One embodiment of expression-inhibitory agent is a nucleic acid that isantisense to a nucleic acid comprising SEQ ID NO: 335-376, 600, 607-866,1120-1122. For example, an antisense nucleic acid (e.g. DNA) may beintroduced into cells in vitro, or administered to a subject in vivo, asgene therapy to inhibit cellular expression of nucleic acids comprisingSEQ ID NO: 335-376, 600, 607-866, 1120-1122. Antisense oligonucleotidespreferably comprise a sequence containing from about 17 to about 100nucleotides and more preferably the antisense oligonucleotides comprisefrom about 18 to about 30 nucleotides. Antisense nucleic acids may beprepared from about 10 to about 30 contiguous nucleotides complementaryto a nucleic acid sequence selected from the sequences of SEQ ID NO:335-376, 600, 607-866, 1120-1122.

The antisense nucleic acids are preferably oligonucleotides and mayconsist entirely of deoxyribo-nucleotides, modifieddeoxyribonucleotides, or some combination of both. The antisense nucleicacids can be synthetic oligonucleotides. The oligonucleotides may bechemically modified, if desired, to improve stability and/orselectivity. Since oligonucleotides are susceptible to degradation byintracellular nucleases, the modifications can include, for example, theuse of a sulfur group to replace the free oxygen of the phosphodiesterbond. This modification is called a phosphorothioate linkage.Phosphorothioate antisense oligonucleotides are water soluble,polyanionic, and resistant to endogenous nucleases. In addition, when aphosphorothioate antisense oligonucleotide hybridizes to its TARGETsite, the RNA-DNA duplex activates the endogenous enzyme ribonuclease(RNase) H, which cleaves the mRNA component of the hybrid molecule.

In addition, antisense oligonucleotides with phosphoramidite andpolyamide (peptide) linkages can be synthesized. These molecules shouldbe very resistant to nuclease degradation. Furthermore, chemical groupscan be added to the 2′ carbon of the sugar moiety and the 5 carbon (C-5)of pyrimidines to enhance stability and facilitate the binding of theantisense oligonucleotide to its TARGET site. Modifications may include2′-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxyphosphorothioates, modified bases, as well as other modifications knownto those of skill in the art.

Another type of expression-inhibitory agent that reduces the levels ofTARGETS is the ribozyme. Ribozymes are catalytic RNA molecules (RNAenzymes) that have separate catalytic and substrate binding domains. Thesubstrate binding sequence combines by nucleotide complementarity and,possibly, non-hydrogen bond interactions with its TARGET sequence. Thecatalytic portion cleaves the TARGET RNA at a specific site. Thesubstrate domain of a ribozyme can be engineered to direct it to aspecified mRNA sequence. The ribozyme recognizes and then binds a TARGETmRNA through complementary base pairing. Once it is bound to the correctTARGET site, the ribozyme acts enzymatically to cut the TARGET mRNA.Cleavage of the mRNA by a ribozyme destroys its ability to directsynthesis of the corresponding polypeptide. Once the ribozyme hascleaved its TARGET sequence, it is released and can repeatedly bind andcleave at other mRNAs.

Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitisdelta virus, group I intron or RNaseP RNA (in association with an RNAguide sequence) motif or Neurospora VS RNA motif. Ribozymes possessing ahammerhead or hairpin structure are readily prepared since thesecatalytic RNA molecules can be expressed within cells from eukaryoticpromoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9). A ribozymeof the present invention can be expressed in eukaryotic cells from theappropriate DNA vector. If desired, the activity of the ribozyme may beaugmented by its release from the primary transcript by a secondribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21:3249-55).

Ribozymes may be chemically synthesized by combining anoligodeoxyribonucleotide with a ribozyme catalytic domain (20nucleotides) flanked by sequences that hybridize to the TARGET mRNAafter transcription. The oligodeoxyribonucleotide is amplified by usingthe substrate binding sequences as primers. The amplification product iscloned into a eukaryotic expression vector.

Ribozymes are expressed from transcription units inserted into DNA, RNA,or viral vectors. Transcription of the ribozyme sequences are drivenfrom a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymeraseII (pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters will be expressed at high levels in all cells; thelevels of a given pol II promoter in a given cell type will depend onnearby gene regulatory sequences. Prokaryotic RNA polymerase promotersare also used, providing that the prokaryotic RNA polymerase enzyme isexpressed in the appropriate cells (Gao and Huang, (1993) Nucleic AcidsRes. 21:2867-72). It has been demonstrated that ribozymes expressed fromthese promoters can function in mammalian cells (Kashani-Sabet, et al.(1992) Antisense Res. Dev. 2:3-15).

A particularly preferred inhibitory agent is a small interfering RNA(siRNA, preferably shRNA). siRNA, preferably shRNA, mediate thepost-transcriptional process of gene silencing by double stranded RNA(dsRNA) that is homologous in sequence to the silenced RNA. siRNAaccording to the present invention comprises a sense strand of 17-25nucleotides complementary or homologous to a contiguous 17-25 nucleotidesequence selected from the group of sequences described in SEQ ID NO:335-376, 600, 607-866, 1120-1122, preferably from the group of sequencesdescribed in SEQ ID No: 335-376, 600, and an antisense strand of 17-23nucleotides complementary to the sense strand. Exemplary sequences aredescribed as sequences complementary to SEQ ID NO: 1-220, 247-333. Themost preferred siRNA comprises sense and anti-sense strands that are 100per cent complementary to each other and the TARGET polynucleotidesequence. Preferably the siRNA further comprises a loop region linkingthe sense and the antisense strand.

A self-complementing single stranded siRNA molecule polynucleotideaccording to the present invention comprises a sense portion and anantisense portion connected by a loop region linker. Preferably, theloop region sequence is 4-30 nucleotides long, more preferably 5-15nucleotides long and most preferably 12 nucleotides long. In a mostpreferred embodiment the linker sequence is GTTTGCTATAAC (SEQ ID NO:334). Self-complementary single stranded siRNAs form hairpin loops andare more stable than ordinary dsRNA. In addition, they are more easilyproduced from vectors.

Analogous to antisense RNA, the siRNA can be modified to confirmresistance to nucleolytic degradation, or to enhance activity, or toenhance cellular distribution, or to enhance cellular uptake, suchmodifications may consist of modified internucleoside linkages, modifiednucleic acid bases, modified sugars and/or chemical linkage the siRNA toone or more moieties or conjugates. The nucleotide sequences areselected according to siRNA designing rules that give an improvedreduction of the TARGET sequences compared to nucleotide sequences thatdo not comply with these siRNA designing rules (For a discussion ofthese rules and examples of the preparation of siRNA, WO2004094636,published Nov. 4, 2004, and UA20030198627, are hereby incorporated byreference).

The present invention also relates to compositions, and methods usingsaid compositions, comprising a DNA expression vector capable ofexpressing a polynucleotide capable of inducing osteoblastdifferentiation and described hereinabove as an expression inhibitionagent.

A special aspect of these compositions and methods relates to thedown-regulation or blocking of the expression of a TARGET polypeptide bythe induced expression of a polynucleotide encoding an intracellularbinding protein that is capable of selectively interacting with theTARGET polypeptide. An intracellular binding protein includes anyprotein capable of selectively interacting, or binding, with thepolypeptide in the cell in which it is expressed and neutralizing thefunction of the polypeptide. Preferably, the intracellular bindingprotein is a neutralizing antibody or a fragment of a neutralizingantibody having binding affinity to an epitope of the TARGET polypeptideof SEQ ID NO: 377-599, 601-606, 867-1119, 1123-1133, preferably SEQ IDNO: 377-599, 601-606. More preferably, the intracellular binding proteinis a single chain antibody.

A special embodiment of this composition comprises theexpression-inhibiting agent selected from the group consisting ofantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for SEQ ID NO: 377-418, 601,867-1119, 1123-1133, preferably SEQ ID NO: 377-418, 601, and a smallinterfering RNA (siRNA) that is sufficiently homologous to a portion ofthe polyribonucleotide coding for SEQ ID NO: 377-418, 601, 867-1119,1123-1133, preferably SEQ ID NO: 377-418, 601, such that the siRNAinterferes with the translation of the TARGET polyribonucleotide to theTARGET polypeptide.

The polynucleotide expressing the expression-inhibiting agent ispreferably included within a vector. The polynucleic acid is operablylinked to signals enabling expression of the nucleic acid sequence andis introduced into a cell utilizing, preferably, recombinant vectorconstructs, which will express the antisense nucleic acid once thevector is introduced into the cell. A variety of viral-based systems areavailable, including adenoviral, retroviral, adeno-associated viral,lentiviral, herpes simplex viral or a sendaviral vector systems, and allmay be used to introduce and express polynucleotide sequence for theexpression-inhibiting agents in TARGET cells.

Preferably, the viral vectors used in the methods of the presentinvention are replication defective. Such replication defective vectorswill usually pack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution, partial deletion or addition ofone or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Preferably, the replication defective virus retainsthe sequences of its genome, which are necessary for encapsidating, theviral particles.

In a preferred embodiment, the viral element is derived from anadenovirus. Preferably, the vehicle includes an adenoviral vectorpackaged into an adenoviral capsid, or a functional part, derivative,and/or analogue thereof. Adenovirus biology is also comparatively wellknown on the molecular level. Many tools for adenoviral vectors havebeen and continue to be developed, thus making an adenoviral capsid apreferred vehicle for incorporating in a library of the invention. Anadenovirus is capable of infecting a wide variety of cells. However,different adenoviral serotypes have different preferences for cells. Tocombine and widen the TARGET cell population that an adenoviral capsidof the invention can enter in a preferred embodiment, the vehicleincludes adenoviral fiber proteins from at least two adenoviruses.Preferred adenoviral fiber protein sequences are serotype 17, 45 and 51.Techniques or construction and expression of these chimeric vectors aredisclosed in US Published Patent Applications 20030180258 and20040071660, hereby incorporated by reference.

In a preferred embodiment, the nucleic acid derived from an adenovirusincludes the nucleic acid encoding an adenoviral late protein or afunctional part, derivative, and/or analogue thereof. An adenoviral lateprotein, for instance an adenoviral fiber protein, may be favorably usedto TARGET the vehicle to a certain cell or to induce enhanced deliveryof the vehicle to the cell. Preferably, the nucleic acid derived from anadenovirus encodes for essentially all adenoviral late proteins,enabling the formation of entire adenoviral capsids or functional parts,analogues, and/or derivatives thereof. Preferably, the nucleic acidderived from an adenovirus includes the nucleic acid encoding adenovirusE2A or a functional part, derivative, and/or analogue thereof.Preferably, the nucleic acid derived from an adenovirus includes thenucleic acid encoding at least one E4-region protein or a functionalpart, derivative, and/or analogue thereof, which facilitates, at leastin part, replication of an adenoviral derived nucleic acid in a cell.The adenoviral vectors used in the examples of this application areexemplary of the vectors useful in the present method of treatmentinvention.

Certain embodiments of the present invention use retroviral vectorsystems. Retroviruses are integrating viruses that infect dividingcells, and their construction is known in the art. Retroviral vectorscan be constructed from different types of retrovirus, such as, MoMuLV(“murine Moloney leukemia virus” MSV (“murine Moloney sarcoma virus”),HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Roussarcoma virus”) and Friend virus. Lentiviral vector systems may also beused in the practice of the present invention.

In other embodiments of the present invention, adeno-associated viruses(“AAV”) are utilized. The AAV viruses are DNA viruses of relativelysmall size that integrate, in a stable and site-specific manner, intothe genome of the infected cells. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies.

In the vector construction, the polynucleotide agents of the presentinvention may be linked to one or more regulatory regions. Selection ofthe appropriate regulatory region or regions is a routine matter, withinthe level of ordinary skill in the art. Regulatory regions includepromoters, and may include enhancers, suppressors, etc.

Promoters that may be used in the expression vectors of the presentinvention include both constitutive promoters and regulated (inducible)promoters. The promoters may be prokaryotic or eukaryotic depending onthe host. Among the prokaryotic (including bacteriophage) promotersuseful for practice of this invention are lac, lacZ, T3, T7, lambdaP_(r), P_(l), and trp promoters. Among the eukaryotic (including viral)promoters useful for practice of this invention are ubiquitous promoters(e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters(e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters(e.g. MDR type, CFTR, factor VIII), tissue-specific promoters (e.g.actin promoter in smooth muscle cells, or Flt and Flk promoters activein endothelial cells), including animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells (Swift, et al. (1984) Cell 38:639-46; Omitz, et al. (1986)Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, (1987)Hepatology 7:425-515); insulin gene control region which is active inpancreatic beta cells (Hanahan, (1985) Nature 315:115-22),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mousemammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95),albumin gene control region which is active in liver (Pinkert, et al.(1987) Genes and Devel. 1:268-76), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf, et al. (1985) Mol. Cell. Biol.,5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1-antitrypsingene control region which is active in the liver (Kelsey, et al. (1987)Genes and Devel., 1: 161-71), beta-globin gene control region which isactive in myeloid cells (Mogram, et al. (1985) Nature 315:338-40;Kollias, et al. (1986) Cell 46:89-94), myelin basic protein gene controlregion which is active in oligodendrocyte cells in the brain (Readhead,et al. (1987) Cell 48:703-12), myosin light chain-2 gene control regionwhich is active in skeletal muscle (Sani, (1985) Nature 314.283-6), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason, et al. (1986) Science 234:1372-8).

Other promoters which may be used in the practice of the inventioninclude promoters which are preferentially activated in dividing cells,promoters which respond to a stimulus (e.g. steroid hormone receptor,retinoic acid receptor), tetracycline-regulated transcriptionalmodulators, cytomegalovirus immediate-early, retroviral LTR,metallothionein, SV40, E1a, and MLP promoters.

Additional vector systems include the non-viral systems that facilitateintroduction of polynucleotide agents into a patient. For example, a DNAvector encoding a desired sequence can be introduced in vivo bylipofection. Synthetic cationic lipids designed to limit thedifficulties encountered with liposome-mediated transfection can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner, et. al. (1987) Proc. Natl. Acad Sci. USA 84:7413-7);see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer,et al. (1993) Science 259:1745-8). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold, (1989) Nature 337:387-8). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO 95/18863 and WO 96/17823, and inU.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenousgenes into the specific organs in vivo has certain practical advantagesand directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, forexample, pancreas, liver, kidney, and the brain. Lipids may bechemically coupled to other molecules for the purpose of targeting.Targeted peptides, e.g., hormones or neurotransmitters, and proteins forexample, antibodies, or non-peptide molecules could be coupled toliposomes chemically. Other molecules are also useful for facilitatingtransfection of a nucleic acid in vivo, for example, a cationicoligopeptide (e.g., International Patent Publication WO 95/21931),peptides derived from DNA binding proteins (e.g., International PatentPublication WO 96/25508), or a cationic polymer (e.g., InternationalPatent Publication WO 95/21931).

It is also possible to introduce a DNA vector in vivo as a naked DNAplasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). NakedDNA vectors for therapeutic purposes can be introduced into the desiredhost cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem.267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al.Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30).Receptor-mediated DNA delivery approaches can also be used (Curiel, etal. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem.262:4429-32).

The present invention also provides biologically compatible, boneformation-enhancing compositions comprising an effective amount of oneor more compounds identified as TARGET inhibitors, and/or theexpression-inhibiting agents as described hereinabove.

A biologically compatible composition is a composition, that may besolid, liquid, gel, or other form, in which the compound,polynucleotide, vector, and antibody of the invention is maintained inan active form, e.g., in a form able to effect a biological activity.For example, a compound of the invention would have inverse agonist orantagonist activity on the TARGET; a nucleic acid would be able toreplicate, translate a message, or hybridize to a complementary mRNA ofa TARGET; a vector would be able to transfect a TARGET cell andexpression the antisense, antibody, ribozyme or siRNA as describedhereinabove; an antibody would bind a TARGET polypeptide domain.

A preferred biologically compatible composition is an aqueous solutionthat is buffered using, e.g., Tris, phosphate, or HEPES buffer,containing salt ions. Usually the concentration of salt ions will besimilar to physiological levels. Biologically compatible solutions mayinclude stabilizing agents and preservatives. In a more preferredembodiment, the biocompatible composition is a pharmaceuticallyacceptable composition. Such compositions can be formulated foradministration by topical, oral, parenteral, intranasal, subcutaneous,and intraocular, routes. Parenteral administration is meant to includeintravenous injection, intramuscular injection, intraarterial injectionor infusion techniques. The composition may be administered parenterallyin dosage unit formulations containing standard, well-known non-toxicphysiologically acceptable carriers, adjuvants and vehicles as desired.

A particularly preferred embodiment of the present composition inventionis a bone formation-enhancing pharmaceutical composition comprising atherapeutically effective amount of an expression-inhibiting agent asdescribed hereinabove, in admixture with a pharmaceutically acceptablecarrier. Another preferred embodiment is a pharmaceutical compositionfor the treatment or prevention of a condition a systemic or localdecrease in mean bone density, or a susceptibility to the condition,comprising an effective bone formation-enhancing amount of a TARGETantagonist or inverse agonist, its pharmaceutically acceptable salts,hydrates, solvates, or prodrugs thereof in admixture with apharmaceutically acceptable carrier.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient. Pharmaceutical compositions for oral usecan be prepared by combining active compounds with solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethyl-cellulose; gums including arabic and tragacanth;and proteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate. Dragee cores may be used in conjunction with suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification or to characterizethe quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Preferred sterile injectable preparations can be a solution orsuspension in a non-toxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, bufferedsaline, isotonic saline (e.g. monosodium or disodium phosphate, sodium,potassium; calcium or magnesium chloride, or mixtures of such salts),Ringer's solution, dextrose, water, sterile water, glycerol, ethanol,and combinations thereof 1,3-butanediol and sterile fixed oils areconveniently employed as solvents or suspending media. Any bland fixedoil can be employed including synthetic mono- or di-glycerides. Fattyacids such as oleic acid also find use in the preparation ofinjectables.

The composition medium can also be a hydrogel, which is prepared fromany biocompatible or non-cytotoxic homo- or hetero-polymer, such as ahydrophilic polyacrylic acid polymer that can act as a drug absorbingsponge. Certain of them, such as, in particular, those obtained fromethylene and/or propylene oxide are commercially available. A hydrogelcan be deposited directly onto the surface of the tissue to be treated,for example during surgical intervention.

Embodiments of pharmaceutical compositions of the present inventioncomprise a replication defective recombinant viral vector encoding thepolynucleotide inhibitory agent of the present invention and atransfection enhancer, such as poloxamer. An example of a poloxamer isPoloxamer 407, which is commercially available (BASF, Parsippany, N.J.)and is a non-toxic, biocompatible polyol. A poloxamer impregnated withrecombinant viruses may be deposited directly on the surface of thetissue to be treated, for example during a surgical intervention.Poloxamer possesses essentially the same advantages as hydrogel whilehaving a lower viscosity.

The active expression-inhibiting agents may also be entrapped inmicrocapsules prepared, for example, by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980) 16th edition, Osol, A. Ed.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™. (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S-S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

As defined above, therapeutically effective dose means that amount ofprotein, polynucleotide, peptide, or its antibodies, agonists orantagonists, which ameliorate the symptoms or condition. Therapeuticefficacy and toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose therapeutically effective in 50% of the population)and LD50 (the dose lethal to 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50. Pharmaceutical compositions thatexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies is used in formulating a range ofdosage for human use. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The pharmaceutical compositions according to this invention may beadministered to a subject by a variety of methods. They may be addeddirectly to TARGET tissues, complexed with cationic lipids, packagedwithin liposomes, or delivered to TARGET cells by other methods known inthe art. Localized administration to the desired tissues may be done bydirect injection, transdermal absorption, catheter, infusion pump orstent. The DNA, DNA/vehicle complexes, or the recombinant virusparticles are locally administered to the site of treatment. Alternativeroutes of delivery include, but are not limited to, intravenousinjection, intramuscular injection, subcutaneous injection, aerosolinhalation, oral (tablet or pill form), topical, systemic, ocular,intraperitoneal and/or intrathecal delivery. Examples of ribozymedelivery and administration are provided in Sullivan et al. WO 94/02595.

Antibodies according to the invention may be delivered as a bolus only,infused over time or both administered as a bolus and infused over time.Those skilled in the art may employ different formulations forpolynucleotides than for proteins. Similarly, delivery ofpolynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

As discussed hereinabove, recombinant viruses may be used to introduceDNA encoding polynucleotide agents useful in the present invention.Recombinant viruses according to the invention are generally formulatedand administered in the form of doses of between about 10⁴ and about10¹⁴ pfu. In the case of AAVs and adenoviruses, doses of from about 10⁶to about 10¹¹ pfu are preferably used. The term pfu (“plaque-formingunit”) corresponds to the infective power of a suspension of virions andis determined by infecting an appropriate cell culture and measuring thenumber of plaques formed. The techniques for determining the pfu titreof a viral solution are well documented in the prior art.

The present invention also provides methods of enhancing bone formation,which comprise the administration to said subject a therapeuticallyeffective amount of an expression-inhibiting agent of the invention. Afurther aspect of the invention relates to a method of treating orpreventing a disease involving a systemic or local decrease in mean bonedensity, comprising administering to said subject a bone formationenhancing pharmaceutical composition as described herein.

The invention also relates to the use of an agent as described above forthe preparation of a medicament for treating or preventing a diseaseinvolving a systemic or local descrease in mean bone density.

In a preferred embodiment of the present invention the disease isselected from the group consisting of osteoporosis, hypercalcemia ofmalignancy, multiple myelomatosis, hyperparathyroidism, andhyperthyroidism. A special embodiment of this invention is a methodwherein the disease is osteoporosis.

Still another aspect of the invention relates to a method for diagnosinga pathological condition involving a systemic or local decrease in meanbone density or a susceptibility to the condition in a subject,comprising determining the amount of polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 377-418,601, 867-1119, 1123-1133 in a biological sample, and comparing theamount with the amount of the polypeptide in a healthy subject, whereinan increase of the amount of polypeptide compared to the healthy subjectis indicative of the presence of the pathological condition.

Preferably the pathological condition is selected from the groupconsisting of osteoporosis, hypercalcemia of malignancy, multiplemyelomatosis, hyperparathyroidism, and hyperthyroidism. More preferably,the pathological condition is osteoporosis.

The polypeptides or the polynucleotides of the present inventionemployed in the methods described herein may be free in solution,affixed to a solid support, borne on a cell surface, or locatedintracellularly. To perform the methods it is feasible to immobilizeeither the polypeptide of the present invention or the compound tofacilitate separation of complexes from uncomplexed forms of thepolypeptide, as well as to accommodate automation of the assay.Interaction (e.g., binding of) of the polypeptide of the presentinvention with a compound can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and microcentrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows thepolypeptide to be bound to a matrix. For example, the polypeptide of thepresent invention can be “His” tagged, and subsequently adsorbed ontoNi-NTA microtitre plates, or ProtA fusions with the polypeptides of thepresent invention can be adsorbed to IgG, which are then combined withthe cell lysates (e.g., (35)^(S)-labelled) and the candidate compound,and the mixture incubated under conditions favorable for complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the plates are washed to remove any unbound label, and thematrix is immobilized. The amount of radioactivity can be determineddirectly, or in the supernatant after dissociation of the complexes.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of the protein binding to theprotein of the present invention quantitated from the gel using standardelectrophoretic techniques.

Other techniques for immobilizing protein on matrices can also be usedin the method of identifying compounds. For example, either thepolypeptide of the present invention or the compound can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated proteinmolecules of the present invention can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the polypeptides of the presentinvention but which do not interfere with binding of the polypeptide tothe compound can be derivatized to the wells of the plate, and thepolypeptide of the present invention can be trapped in the wells byantibody conjugation. As described above, preparations of a labeledcandidate compound are incubated in the wells of the plate presentingthe polypeptide of the present invention, and the amount of complextrapped in the well can be quantitated.

The polynucleotides of the invention, complementary to SEQ ID NO: 1-220,247-333 increase osteoblast differentiation.

Accordingly, another embodiment of the present invention relates to amethod for in vitro production of bone tissue, comprising the steps ofcontacting undifferentiated mammalian cells with a polynucleotidesequence comprising a sequence selected from the group consisting ofsequences complementary to SEQ ID No: 335-376, 600, 607-866, 1120-1122,preferably selected from the group consisting of sequences complementaryto SEQ ID NO: 1-220, 247-333 for a time sufficient to differentiate theundifferentiated cells into osteoblasts, thereby producing a continuousbone matrix.

In a preferred embodiment, the method comprises the steps of:

-   -   (a) applying undifferentiated mammalian cells on a substrate to        form a cellular substrate,    -   (b) introducing a polynucleotide comprising a nucleotide        sequence selected from the group consisting of sequences        complementary to SEQ ID No: 335-376, 600, 607-866, 1120-1122,        preferably selected from the group consisting of sequences        complementary to SEQ ID NO: 1-220, 247-333, for a time        sufficient to differentiate the undifferentiated cells into        osteoblasts, thereby producing a continuous bone matrix.

The invention thus provides a method for producing a substrate with amatrix grown thereon, which matrix may be used for the provision ofload-bearing implants, including joint prostheses, such as artificialhip joints, knee joints and finger joints, and maxillofacial implants,such as dental implants. It can also be used for special surgerydevices, such as spacers, or bone fillers, and for use in augmentation,obliteration or reconstitution of bone defects and damaged or lost bone.Bone formation can be optimized by variation in mineralization, both byinductive and by conductive processes.

The present invention also relates to a combination of a load-bearingimplant (preferably coated with a matrix as described above) with a bonefiller comprising a matrix as described.

The method of the invention is also very suitable in relation torevision surgery, i.e., when previous surgical devices requirereplacement.

Suitable undifferentiated cells are bone marrow cells, includinghaematopoietic cells and in particular stromal cells. The marrow cells,and especially the stromal cells are found to be very effective in thebone producing process when taken from their original environment.

The undifferentiated cells can be directly applied on the substrate orthey can advantageously be multiplied in the absence of the substratebefore being applied on the substrate. In the latter mode, the cells arestill largely undifferentiated after multiplication and, for the purposeof the invention, they are still referred to as undifferentiated.Subsequently, the cells are allowed to differentiate. Differentiationcan be induced or enhanced by the presence of suitable inductors, suchas glucocorticoids, and dexamethasone. Suitable inductors ofdifferentiation are the expression inhibitory agents of the presentinvention.

The use of undifferentiated cells provides several advantages. Firstly,their lower differentiation implies a higher proliferation rate andallows the eventual functionality to be better directed and controlled.Moreover, culturing these cells not only produces the required bonematrix containing organic and inorganic components, but also results inthe presence, in the culture medium and in the matrix, of severalfactors which are essential for growth of the tissue and for adaptationto existing living tissue. Also, the culture medium can be a source ofactive factors such as growth factors, to be used in connection with theimplanting process. Furthermore, such undifferentiated cells are oftenavailable in large quantities and more conveniently than e.g., maturebone cells, and exhibit a lower morbidity during recovery. Moreover, theundifferentiated cells can be obtained from the patient for whom theimplant is intended. The bone resulting from these cells is autologousto the patient and thus no immune response will be induced. Matrices asthick as 100 μm can be produced as a result of the use ofundifferentiated cells.

The substrate on which the undifferentiated cells can be applied andcultured can be a metal, such as titanium, cobalt/chromium alloy orstainless steel, a bioactive surface such as a calcium phosphate,polymer surfaces such as polyethylene, and the like. Although lesspreferred, siliceous material such as glass ceramics, can also be usedas a substrate. Most preferred are metals, such as titanium, and calciumphosphates, even though calcium phosphate is not an indispensablecomponent of the substrate. The substrate may be porous or non-porous.The cells can be applied at a rate of e.g., 10³-10⁶ per cm², inparticular 10⁴-2×10⁵ cells per cm².

The culture medium to be used in the method according to the inventioncan be a commonly known culture medium such as MEM (minimum essentialmedium). Advantageously, the medium can be a conditioned medium. In thiscontext, a conditioned medium is understood to be a medium whereinsimilar cells have previously been incubated, causing the medium tocontain factors such as polypeptides, secreted by the cells which areimportant for cell growth and cell differentiation.

The cells are cultured for a time sufficient to produce a matrix layer,e.g., a matrix layer having a thickness of at least 0.5 μm, inparticular from 1 up to 100 μm, more in particular of 10-50 μm. Thecells may be contacted with the culture medium for e.g. 2-15 weeks, inparticular 4-10 weeks.

The production of the matrix, when applied on a substrate, results in acontinuous or quasi-continuous coating covering the substrate for atleast 50%, in particular at least 80% of its surface area.

The present invention further relates to the osteoblast cells obtainableby the above method.

Still another aspect or the invention relates to a method for diagnosinga pathological condition involving cognitive impairment or asusceptibility to the condition in a subject, comprising determining theamount of polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 377-418, 601, 867-1119, 1123-1133 ina biological sample, and comparing the amount with the amount of thepolypeptide in a healthy subject, wherein an increase of the amount ofpolypeptide compared to the healthy subject is indicative of thepresence of the pathological condition.

The invention is further illustrated in the following figures andexamples.

Experimental Section

EXAMPLE 1 Development of a High-Throughput Screening Method for theDetection of Endogenous Alkaline Phosphatase

Adenoviral Controls.

Ad-BMP2: described in WO 03/018799

Ad-eGFP: Referred to as pIPspAdApt6-EGFP in WO 02070744

Ad-hCAR: hCAR cDNA is isolated using a PCR methodology. The hCAR cDNA isPCR amplified from a HeLa cell cDNA library (Quick clone, Clontech). Asingle fragment of 1119 bp is obtained and digested with the HindIII andBamHI restriction enzymes. pIPspAdapt6 vector (WO99/64582) is digestedwith the same enzymes, gel-purified and used to ligate to the digestedPCR hCAR fragment. AdC15 (Ad5/Ad35) and AdC20 (Ad5/Ad51) viruses aregenerated as described in WO02/24933

Ad5-luc_v13: Cloned by PCR and virus generated as described in WO03020931

Ad5-M6PR_v1: Cloned using Sap1-sites into vector and virus generated asdescribed in WO03/020931.

H9: Cloned using Sap1-sites into vector and virus generated as describedin WO03/020931

H11: Cloned using Sap1-sites into vector and virus generated asdescribed in WO03/020931

Principle of the Assay

Mesenchymal progenitor cells (MPCs) differentiate into osteoblasts inthe presence of appropriate factors (e.g. BMP2). An assay to screen forsuch factors is developed by monitoring the activity of alkalinephosphatase (AP) enzyme, an early marker in the osteoblastdifferentiation program. MPCs are seeded in 384 well plates andsimultaneously co-infected one day later with adenoviruses encoding thehuman coxsackie and adenovirus receptor (hCAR; Ad-hCAR) and individualsiRNA adenoviruses (Ad-siRNA) from the SILENCESELCET® collection.AdC15-hCAR/ AdC20-hCAR co-infection increases the AdC01-siRNA infectionefficiency. Cellular AP activity is determined 13 days after the startof the infection (13 dpi). (FIG. 2 illustrates the principle of theassay).

Development of the Assay

MPCs are isolated from the bone marrow of healthy volunteers, obtainedafter informed consent (Cambrex/Biowhittaker, Verviers, Belgium).

In a series of experiments, carried out in 384 well plates, severalparameters are optimized: cell seeding density, multiplicities ofinfection (MOI) of control viruses (Ad-BMP2 or Ad-eGFP), MOI of Ad-hCAR,duration of infection, toxicity, infection efficiency (using Ad-eGFP)and the day of readout.

Using Ad-BMP2 (BMP2 overexpression) as a positive control for assaydevelopment, the following protocol results in the highest dynamic rangefor the assay with the lowest standard deviation on the backgroundsignal:

MPCs are seeded on day 0 at 500 cells per well of a 384 well plate andco-infected the next day using a mix of Ad-hCAR (5 μl of an Ad-hCARsolution: mix total MOI=155.7) and 1 μl of Ad-control-virus (Ad-BMP2 orAd-eGFP; corresponds to a theoretical MOI of 5000). On day 5, the mediumcontaining the virus is removed and replaced by fresh medium containingno virus. Up-regulation of alkaline phosphatase is read at 13 dpi (dayspost infection): 15 μl 4-Methylumbelliferylphosphate (MUP, Sigma) isadded to each well, the plates are incubated for 15 min at 37° C. andmonitored for AP activity using a fluorescence plate reader (Fluostar,BMG).

After optimisation of the assay, a small pilot screen is run (103different Ad-siRNA viruses) with the use of robotics (96/384 channeldispensor Tecan Freedom 200 equipped with TeMO96, TeMO384 and RoMa,Tecan AG, Switzerland). The TARGETS from this screen are collected andretested in the same assay. The two Ad-siRNAs that score strongest(H9=H24-010; H10=H24-011) are used to generate a control plate(knock-down (KD) control plate) containing Ad-siRNAs. The control plate,a 96 well plate containing 3 negative (N1,N2,N3) and 3 positive(P1,P2,P3) control viruses is depicted in FIG. 3. This “knock-down”control plate contains Ad-H9 (H24-010) and Ad-H 10 (H24-011) as positivecontrols; Ad-eGFP (knock-in virus) as infection control; andAd-eGFP-siRNA, Ad-M6PR-siRNA and Ad-Luc-siRNA (all 3 are knock-downviruses) as negative controls.

The control viruses are pipetted from 96 well KD control plates into 384well plates using robotics. (The final lay-out of the 384 well plate isdepicted in FIG. 4).

FIG. 5 shows the results of the automated screening procedure using theKD control plate. The mean and standard deviations of the KD negativecontrols (N1-N3) are used to calculate a cut-off for TARGET analysis,which is set at the mean for N1, N2, N3 (‘All negatives’) plus 3 timesthe standard deviation for ‘All negatives’. The positive controls (P1and P2), scored in more than 95% of the infected wells. The negativecontrol viruses scored in less than 5% of the wells.

EXAMPLE 2 Screening of 7980 Ad-siRNA Adenoviruses in the OsteogenesisAssay

The optimized protocol for screening the SILENCESELECT® library is thefollowing: on day 0, MPC cells are seeded in black 384 well plates withclear bottom (Costar or Nunc) in 60 μl medium at a density of 500 cellsper well. One day later, 1 μl Ad-siRNA virus from the SILENCESELECT®collection, stored in 384 well plates (estimated titer of 2.5×10⁹ viralparticles per ml) and 5 μl of Ad-hCAR solution (total MOI=155),dispensed in 96 well V-bottom plates, is transferred with the aid of a96/384 channel dispenser (Tecan Freedom 200 equipped with TeMO96,TeMO384 and RbMa, Tecan AG, Switzerland) from the wells of a 96 wellplate containing the Ad-hCAR solution to each of the wells of the 384well plates containing MPCs. The KD control plate is run under the sameconditions as the aliquot plates from the SILENCESELEST® collection. AllAd-siRNA viruses are screened in duplicate, with each singular on adifferent MPC plate. Plates are then incubated at 37° C. Four days postinfection the medium containing the adenoviruses is replaced by freshmedium free of virus. Thirteen days post infection, the AP activityreadout is performed. A typical result of a 384 well screening plate isdepicted in FIG. 6, in which the relative fluorescence units (RFU) areplotted for each of the data points of the 384 well plate on the Y-axis;while the numbers on the X-axis correspond to positions in the 384 wellplate.

This duplicate screen is done twice, and all four data points are usedfor TARGET identification (see Example 3).

EXAMPLE 3 Target Identification Using the AP Assay

The data obtained from measuring the AP activity in Examples 1 and 2 areanalyzed as follows: the background is calculated by taking the mean ofthe data points from all the plates except the control plate. A cut-offvalue for TARGET identification is calculated by adding 3 times thestandard deviation of all data points, excluding the control plate. Eachdata point is analyzed for scoring above or under the cut-off. OnlyAd-siRNAs inducing endogenous AP activity levels above the cut-off areof further interest. TARGETS are prioritized according to their scoringin single or duplicate, in one or both of the screens. Data arecollected for 7980 Ad-siRNA virus constructs representing 4091independent genes. An overview of the constructs is given in Table 1.

One of the identified hits has been shown to be a bone anabolic factorbefore and therefore validates the assay:

H24-034: SRC

Marzia et al. (2000) showed that bone formation was increased in Srcnull mice compared to wild-type mice. Most relevant to this work,osteoblasts isolated from Src null mice or osteoblasts isolated fromwild-type mice but transfected with Src-antisense oligonucleotidesshowed increased AP activity in vitro.

8 genes identified in the screen were targeted by 2 Ad-siRNAs. Thesegenes are AVPR1B, FLJ22955, IL1F8, PPIA, USP38, C9, LOC254378 and BRS3(see Table 1).

The siRNA sequences of the present invention comprise sequencescomplementary to sequences corresponding to the identified KD TARGETS(SEQ ID NO: 1-220, 247-333). For the preferred TARGETS, thecorresponding polynucleotides known in public databases (referred to asSEQ ID NO: 335-376, 600, 607-866, 1120-1122), their respective names andthe polypeptides translated from those polynucleotides (referred to asSEQ ID NO: 377-418, 601, 867-1119, 1123-1133), are listed in Table 1.

EXAMPLE 4 Quality Control of the Target Ad-siRNAs

The Ad-siRNA TARGETS are subjected to further analysis to establishtheir therapeutic application as bone anabolic factors. The Ad-siRNA issubjected to quality control analysis (this example). Other validationsteps are the validation of the TARGETS at the mRNA level (Example 5),screening of the targets in osteogenesis assays such as themineralization assay (Example 6), and development of additionalAd-siRNAs targeting the identified genes (Example 9). TARGETS thatremain of interest after these validation assays are considered for drugdiscovery and assays are developed allowing the discovery andoptimization of compounds that mimick the bone anabolic actions of thetarget Ad-siRNAs (Example 7). In addition, the anti-resorptiveactivities of the identified Ad-siRNAs are validated in osteoclastassays (Example 8).

Verifying the Identity of the siRNA Insert from the TARGET Ad-siRNAs

TARGET Ad-siRNAs are propagated using PERC6/E2A cells (see WO99/64582)seeded in a 96-well plate, followed by re-screening of these viruses atseveral MOI's in the primary assay (see Example 1) and by sequencing thesiRNAs encoded by the TARGET Ad-siRNA viruses. This procedure is carriedout as follows.

PERC6/E2A cells are seeded in 96-well plates at a density of 40,000cells/well in 180 μl PERC6/E2A medium. Cells are then incubatedovernight at 39° C. in a 10% CO₂ humidified incubator. One day later,cells are infected with 1 μl of crude cell lysate from SILENCESELECT®stocks containing TARGET Ad-siRNAs. Cells are further incubated at 34°C., 10% CO₂ until appearance of cytopathic effect (cpe, as revealed bythe swelling and rounding up of the cells which typically occurs 7 dayspost infection). The supernatant is collected and the virus crude lysateis treated with proteinase K: 12 μl crude lysate is added to 4 μl Lysisbuffer (1× Expand High Fidelity buffer with MgCl₂ (Roche MolecularBiochemicals, Cat. No 1332465) supplemented with 1 mg/ml proteinase K(Roche Molecular Biochemicals, Cat No 745 723) and 0.45% Tween-20 (RocheMolecular Biochemicals, Cat No 1335465) in sterile PCR tubes. These areincubated at 55° C. for 2 h followed by a 15 min inactivation step at95° C. For the PCR reaction, 1 μl lysate is added to a PCR master mixcomposed of 5 μl 10× Expand High Fidelity buffer with MgCl₂, 0.5 μl ofdNTP mix (10 mM for each dNTP), 1 μl of ‘Forward primer’ (10 mM stock,sequence: 5′ CCG TTT ACG TGG AGA CTC GCC, SEQ ID NO: 245) and 1 μl‘Reverse Primer’ (10 mM stock, sequence: 5′ CCC CCA CCT TAT ATA TAT TCTTTC C, SEQ ID NO: 246), 0.2 μl of Expand High Fidelity DNA polymerase(3.5 U/μl, Roche Molecular Biochemicals) and 41.3 μl H₂O. PCR isperformed in a PE Biosystems GeneAmp PCR system 9700 as follows: the PCRmixture (50 μl in total) is incubated at 95° C. for 5 min; each cycleruns at 95° C. for 15 sec, 55° C. for 30 sec, 68° C. for 4 min, and isrepeated for 35 cycles. A final incubation at 68° C. is performed for 7min. 5 μl of the PCR mixture is mixed with 2 μl of 6× gel loadingbuffer, loaded on a 0.8% agarose gel containing 0.5 μg/μl ethidiumbromide to resolve the amplification products. The size of the amplifiedfragments is estimated from a standard DNA ladder loaded on the samegel. The expected size is ˜500 bp.

For sequencing analysis, the siRNA constructs expressed by the TARGETadenoviruses are amplified by PCR using primers complementary to vectorsequences flanking the SapI site of the pIPspAdapt6-U6 plasmid. Thesequences of the PCR fragments are determined and compared with theexpected sequence.

For the KD TARGET sequences identified (Table 1), sequence analysisconfirmed that the siRNA present in the TARGET Ad-siRNA had the expectedsequence.

Multiple MOI Rescreen

The propagated TARGET Ad-siRNAs were rescreened at several MOIs in theAP assay (Example 1). The Ad-siRNAs had to score in duplo at at leastone MOI to pass this quality control step.

All TARGETS listed in Table 1 fulfilled this quality control step andthus:

a) showed the correct length of the PCR fragment

b) showed the correct sequence of the PCR fragment

c) induced AP activity in duplicate for at least 1 MOI.

EXAMPLE 5 mRNA Validation Experiments for Identified Targets

A validation of the target Ad-siRNAs is carried out on RNA isolated frominfected MPCs. First, the expression of the targets is analyzed inseveral isolates of primary human MPCs and osteoblasts (hOBs). Second,the knock-down of the target gene expression by the Ad-siRNA is verifiedat the mRNA level. Third, the upregulation of endogenous bone AP mRNAversus that of placental or intestinal AP mRNA is analyzed.

MPC and Osteoblast Expression Analysis for the Identified TargetsProfiling

Expression levels of target genes are determined in 4 different isolatesof MPCs and 2 different isolates of hOBs. The MPCs and hOBs (obtainedfrom Cambrex/Biowhittaker, Verviers, Belgium) are seeded at 3000 resp.5000 cells/cm² in T180 flasks and cultured until they reach 80%confluency. The cells are washed with ice-cold PBS and harvested byadding 1050 μl SV RNA Lysis Buffer to a T180 flask. Total RNA isprepared using the SV Total RNA isolation System (Promega, Cat #Z3100).The concentration of the total RNA is measured with the Ribogreen RNAQuantification kit (Molecular Probes, Leiden, The Netherlands, Cat No.R-11490). cDNA synthesis is performed using 40 ng total RNA per reactionusing the TaqMan Universal PCR Master Mix, No AmpErase UNG, kit (AppliedBiosystems, Warrington, UK,Part number 4324018). For each RT reaction aminus-RT reaction (negative control: no enzyme included in the reaction)is performed.

The real-time reverse transcriptase (rtRT)-PCR reaction is performedwith gene specific primers on both cDNA and minus-RT samples, using theSYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK, Partnumber 4309155). For the normalization of the expression levels, aRT-PCR reaction is performed on human β-actin using the Human β-actinkit (Applied Biosystems, Warrington, UK, Part number 4310881E). Thefollowing program is run on a real-time PCR apparatus (ABI PRISM 7000Sequence Detection System): 10 min at 25° C., 30 min at 48° C., 5 min at95° C. In FIGS. 7A and 8, relative expression levels for 10 genes aredepicted for respectively MPC and hOB isolates. For the data in FIG. 7A,total RNA is extracted from 4 different MPC isolates and used to analyzeexpression levels of target genes identified through the targetAd-siRNAs. RtRT-PCR compatible primer sets (Table 4) are developed for10 genes and compared to expression levels of β-actin. Data areexpressed as −log(difference to β-actin) (Y-axis). For the datapresented in FIG. 8, total RNA is extracted from 2 different hOBisolates.

TABLE 4 SEQ ID Gene Primer Name Sequence NO. CALCRL CALCRL_RevAGAGACCAAAAGACCCTGGAAGT 222 SRC SRC_For ACAGCGGCGGCTTCTACA 223 SRCSRC_Rev CATCGGCGTGTTTGGAGTAGT 224 PSMB3 PSMB3_For ATCCGGATCACCTGTTTGAAAC225 PSMB3 PSMB3_Rev GTGGTGATTTTGTCCTTCTCGAT 226 HP43.8KD HP43.8KD_ForCCATACACAGAGGGAAGCATACG 227 HP43.8KD HP43.8KD_RevCAGTCTTGCTGTGATCTGGGAGTA 228 APEX APEX_For GCATAGGCGATGAGGAGCAT 229 APEXAPEX_Rev GACCTCGGCCTGCATTAGG 230 GPR38 GPR38_For CATCGTCGCTCTGCAACTTTT231 GPR38 GPR38_Rev CCGCTCTGTACTTCTTTGAAATGA 232 ROCK2 ROCK2_ForCCTGGTGGAGACCTTGTAAACCT 233 ROCK2 ROCK2_Rev AGCAAGAACAACTTCAGCAGTGTAA234 CXCR6 CXCR6_For GCCATGACCAGCTTTCACTACA 235 CXCR6 CXCR6_RevGTTAAGGCAGGCCCTCAGGTA 236 OPN3 OPN3_For CTAACCGTGCTGGCCTATGAAC 237 OPN3OPN3_Rev CAGGCCCAGGAAAAATTGATC 238 FUK FUK_For TTCGCGATCAGCCCCTTAC 239FUK FUK_Rev ACTCACTGGCTGAGGAGGTCAT 240

Analysis of the Knock-down of the Target Gene Expression by the Ad-siRNAis Verified at the mRNA Level.

To determine whether the target Ad-siRNAs result in knock-down ofexpression from the corresponding gene, total RNA is harvested fromAd-siRNA infected MPCs and gene expression is analyzed usinggene-specific primers.

MPCs are seeded at 25,000 cells per well of a 24 well plate. After 24hours the cells are infected with knock-down hit viruses or negativecontrol viruses Ad-gPPARg and Ad-GL2.2 that knock down the expression ofrespectively PPARγ (all four known splice variants) and luciferase, bothof which are not related to osteogenesis. For Ad-siRNAs, cells areco-infected with Ad-hCAR and Ad-siRNA crude lysates (MOI Ad-hCAR: 750;40, 10, 3.3 μL virus with average titer of 2.5×10E9 virus particles/ml).At 5 dpi the medium is refreshed and at 14 dpi the cell lysates areprepared. Cells are processed for mRNA analysis as described in theprevious section. mRNA levels for a specific gene are normalized forβ-actin levels and compared to levels measured for the negative controlAd-siRNAs. An example of these kind of analyses is provided in FIG. 7B.Data are normalized for β-actin expression levels and compared toendogenous gene expression for MPCs infected with negative controlviruses (Y-axis: percent gene expression of endogenous gene;100%=endogenous mRNA levels present in negative control sample).

Analysis of the Upregulation of Endogenous Bone AP mRNA Versus that ofPlacental or Intestinal AP mRNA.

BAP is the physiologically relevant AP involved in bone formation. Inorder to determine whether the measured AP activities are due toupregulation of BAP expression or of another AP gene product, mRNAlevels for all AP genes are analysed for infected MPCs. mRNA levels aredetermined as described in the previous sections. The difference is inthe primer set used (see Table 5): one set detects BAP ALPL (humanalkaline phosphatase liver/bone/kidney) mRNA expression. Another setdetects the expression of the 3 other AP genes (ALPI (human alkalinephosphatase intestinal), ALPP (human alkaline phosphatase placental(PLAP)), and ALPPL2 (human alkaline phosphatase placental-like)). ALPI,ALPP and ALPPL2 are highly similar at the nucleotide level and cantherefore be amplified using one primer pair.

The primer pairs are first validated on RNA isolated from MPCs infectedwith Ad-eGFP and Ad-BMP2. FIG. 7 illustrates the strong upregulation ofBAP mRNA by Ad-BMP2 and the absence of upregulation of expression of anyof the other AP genes. MPCs are infected in 24 well plate format usingAd-eGFP (negative control) or the osteogenic Ad-BMP2. Cells areharvested and RNA is prepared and subjected to rtRT-PCR using primersets amplifying BAP mRNA or mRNA from the other 3 AP genes (PLAP/IAP).Ad-BMP2 strongly upregulates BAP mRNA levels but not the mRNA levels ofthe other 3 AP genes.

Both primer sets are then used to measure mRNA levels for all AP genesin RNA isolated from Ad-siRNA infected MPCs.

TABLE 5 SEQ ID Name Sequence NO: JDO-05F (PLAP) TTCCAGACCATTGGCTTGAGT241 JDO-05bisR ACTCCCACTGACTTTCCTGCT 242 (PLAP/ALPI/ALPPL2) JDO-21F(BAP) CATGCTGAGTGACACAGACAAGAAG 243 JDO-21R (BAP)TGGTAGTTGTTGTGAGCATAGTCCA 244

EXAMPLE 6 Mineralization

The process of osteogenesis consists of several successive events.During the initial phases of osteogenesis, bone alkaline phosphatase(BAP) becomes upregulated. It is however equally important to look atspecific events occurring in later stages of osteogenesis such asmineralization. During differentiation, cells deposit (hydroxy)apatite(Ca²⁺-phosphate precipitate) on an extracellular matrix consistingmostly of collagen type I to form mineralized bone.

Assay Setup

In the bone cell mineralizing assay (BM assay), primary human MSCs aredifferentiated in vitro into mineralizing osteoblasts using BMP2(recombinant or delivered by adenoviral transduction) as an osteogenicagent. Mineralization is then visualized by staining the MSCs withAlizarin Red, a dye with a high affinity for calcium (see FIG. 8).

Screening and TARGET Identification

The following optimized protocol is used for screening Ad-siRNA andAd-cDNA TARGETS identified in the primary assay:

100,000 MPCs are seeded in each well of a 6 well plate in 2 ml MSCmedium, containing 10% FCS. The next day, after incubation at 37° C.,10% CO₂ in a humidified incubator, cells are co-infected with AdCI5-hCAR(final MOI of 750) and Ad-siRNA, Ad-cDNA or control viruses at a finalMOI of 1250, 2500 and 5000. Cells are incubated at 37° C., 10% CO₂ in ahumidified incubator for a further six days. Virus is removed andreplaced by 2 ml fresh MSC medium, 10% FCS. Over the next 22 days,medium is refreshed 3 times in 2 weeks. Every other time, medium isrefreshed half or completely. At 28 days after the start of theexperiment, the conditioned medium is removed, cells are fixed using 10%paraformaldehyde and the monolayers stained with 1 mL of ˜1% AlizarinRed (Sigma, #A5533) in MilliQ water (pH adjusted to 4.2). Ad-eGFP, toassess infection efficiency, Ad-BMP2 as strong osteogenic inducer andAd-H4-2 as a weak osteogenic factor are included in each experiment asnegative and positive controls, respectively. Every experiment whereAd-H4-2 did not induce mineralization is entirely repeated.

The KD TARGET sequences of Ad-shRNAs that induced mineralization arepresented in Table 6.

TABLE 6 KD TARGET SEQ Genbank TARGET Sequence ID TARGET DNA/RNA Id(5′→3′) NO. TARGET Gene Name Gene Symbol Accession H24-001 CAACTTGTACCTG3 G protein-coupled GPR38 NM_001507 GGCAGC receptor 38 H24-004CATGCTGTTTGAG 6 MAP kinase-interacting MKNK2- NM_017572- AGCATCserine/threonine kinase 2 MNK2 SK236 H24-006 GACGGTGTTAATG 8 caseinkinase 1, gamma 1 CSNK1G1- NM_022048- ATAGCC CK1g1 SK647 H24-007CTTCGGCACTCCT 9 myosin light chain kinase HSA247087- AJ247087- GAGTTCcaMLCK SK536 H24-009 ACGCAAAGTGGC 11 tRNA IPT NM_017646 CAGGAGCisopentenyltransferase 1 H24-013 CAACCTGCTGGTG 15 opsin 3(encephalopsin, OPN3 NM_014322 CTCGTC panopsin) H24-014 CTCTCTTAGATCT 16granzyme K (serine GZMK NM_002104 GGAACC protease, granzyme 3; tryptaseII) H24-015 AGCAGGAAGGCG 17 ubiquitin-specific pro- AF073344- AF073344-GACATAC tease 3 - ubiquitin USP3 NM_006537 specific protease 3 H24-018TCAGGTAGTTGGT 20 coagulation factor XIII, F13A1 NM_000129 TCTGAC A1polypeptide H24-019 CTGCGCCGAACA 21 proteasome (prosome, PSMB3 NM_002795AATGTAC macropain) subunit, beta type, 3 H24-020 TGTGGCGACTTGT 22 ClpXcaseinolytic CLPX NM_006660 GCACAC protease X homolog (E. coli) H24-021TCTCTCAGTGTAG 23 hypothetical protein FLJ14906 NM_032859 AATGCC FLJ14906H24-024 GTGTACTGGTACA 26 matrix metalloproteinase MMP23A- NM_004659-AGGACC 23A - matrix MMP23B NM_006983 metalloproteinase 23B H24-026TCTCTCATCAATA 28 APEX nuclease APEX NM_001641- CTGGTC (multifunctionalDNA NM_080648- repair enzyme) NM_080649 H24-029 CTATGCCATCACC 31LOC254378 LOC254378 XM_174812 TTCTGC H24-030 TGTGCCGAAGGAT 32 similar toa disintegrin LOC137491 XM_070459 GTAAGC and metalloprotease domain 25(testase 2) H24-031 CCGGGACATAACT 33 similar to bile salt- LOC138529XM_070951 AAATCC dependent lipase oncofetal isoform H24-032 AGCAGGCTATGG34 complement component 9 C9 NM_001737 GATCAAC H24-033 CCACAAGGTTGCA 35xylulokinase homolog (H. XYLB NM_005108 GCATTC influenzae) H24-035GGGCTCAGCCAG 36 chaperone, ABC1 activity CABC1- NM_020247- GAGATTC ofbc1 complex like (S. ADCK3 SK609 pombe) H24-036 CAGGTAGACATG 37fyn-related kinase FRK NM_002031 GCGGCAC H24-038 GCACGATTTGGAG 39unc-51-like kinase 1 (C. ULK1 NM_003565 GTCGCC elegans) H24-041GGACTCTCAGTTC 42 phosphoinositide-3- PIK3C2B NM_002646 AGCATC kinase,class 2, beta polypeptide H24-049 GTACCTGCAGGTG 1 arginine vasopressinAVPR1B NM_000707 CTCAGC receptor 1B H24-054 GTACCTGCGGCAG 54 chemokine(C—C motif) CCR1 NM_001295 TTGTTC receptor 1 H24-062 TTCGGACACCCAC 62retinal pigment RRH NM_006583 AAATGC epithelium-derived rhodopsinhomolog H24-064 GTTGTCCTGTTCT 63 olfactory receptor, OR1A2 NM_012352GACGTC family 1, subfamily A, member 2 H24-071 CACCTGCTTTCTC 70 KIAA1453protein KIAA1453 NM_025090 AATGCC H24-073 AGCACCTCGCTGA 72sentrin/SUMO-specific SENP3 NM_015670 CATTCC protease 3 H24-078GCTTCTGGTGGAG 77 transglutaminase 3-like TGM3L XM_066181 AAGGAC H24-079GTGTATGAAGTGG 78 similar to solute carrier LOC160662 XM_090422 TCCACCfamily 21 (organic anion transporter), member 8 H24-084 CAGTGCCAAGAA 83neuron navigator 2 NAV2 NM_018162 GGAGCCC H24-092 ATGCAGGTCCATA 91transient receptor poten- TRPM6 NM_017662 TGTGAC tial cation channel,subfamily M, member 6 H24-093 CCTTTCTCTGAAC 92 ataxia telangiectasia andATR NM_001184 ACGGAC Rad3 related H24-095 CAGGTTCTCCTCA 94 similar toTPA: G-protein LOC126788 XM_060177 AACGGC coupled receptor H24-097ACATCCTGCTGTC 96 thousand and one amino TAO1 - PSK NM_004783- AGAGCCacid protein kinase- NM_016151 prostate derived STE20- like kinase PSKH24-099 GTTCTCCAGTGCC 98 solute carrier family 16 SLC16A3 NM_004207ATTGGC (monocarboxylic acid transporters), member 3 H24-104AGTGCGCATCTTC 103 fibroblast growth factor FGF14 NM_004115 GGCCTC 14H24-106 GCCCTGATGTCCA 105 NADPH-dependent FMN NR1 NM_014434 TCTTCC andFAD containing oxidoreductase H24-107 CATAGGGAAGGA 106 interleukin 1family, IL1F8 NM_014438 CACTTGC member 8 (eta) H24-108 CCTGGATGTGAGA 107interleukin 1 family, IL1F8 NM_014438 GAGAGC member 8 (eta) H24-109AACTTGTACTATG 108 Ras association RASSF2 NM_014737 AAGGCC (RalGDS/AF-6)domain family 2 H24-110 GTATTCTGTACAC 109 adrogen-regulated short-ARSDR1 NM_016026 CCTGGC chain dehydrogenase/reductase 1 H24-111TTCTCGCAATGGC 110 peptidylprolyl isomerase PPIL1 NM_016059 CAATGC(cyclophilin)-like 1 H24-112 GAAGAACAGCAG 111 RAS, dexamethasone- RASD1NM_016084 CCTGGAC induced 1 H24-113 TCAGGCGGATCTT 112dicarbonyl/L-xylulose DCXR NM_016286 GACAGC reductase H24-117TCTCTCCACACAA 116 chromosome 20 open C20orf121 NM_024331 ACCTTC readingframe 121 H24-119 GCGAATTCCACCA 118 solute carrier family 26, SLC26A8NM_052961 GCATTC member 8 H24-120 TGTCCAGGACCTA 119 UDPglycosyltransferase 1 UGT1A1 NM_000463 TTGAGC family, polypeptide A1H24-128 TGTGCGAGACCTC 127 HMT1 hnRNP HRMT1L3 NM_019854 GATTTCmethyltransferase-like 3 (S. cerevisiae) H24-130 AGCATGAAAGAA 129peroxisomal short-chain ENSG00000169066- ENSG00000169066- ACCCTGCalcohol dehydrogenase humNRDR NM_021004 H24-131 GAAGATCACCATT 130similar to peptidylpro- PPIA- NM_021130- GCTGAC lylisomerase A (cyclo-LOC127711- XM_060625- philin A) - similar to LOC128430- XM_066074-Peptidylprolyl cis-trans LOC138130- XM_070771- isomerase A (PPIase)LOC165317- XM_092514- (Rotamase) (Cyclophilin LOC257232 XM_172314 A)(Cyclosporin A- binding protein) (SP18) H24-133 TGCAGGCAAGCA 1323-oxoacid CoA transfer- OXCT2 NM_022120 GACGGTC ase 2 H24-136CTTATTGTTCACA 135 similar to glyceraldehyde LOC170327 XM_093255 TTGGCC3-phosphate dehydrogenase H24-138 TCAGGTGTCCCAT 137 interleukin-1receptor- IRAK2 NM_001570- TCCAGC associated kinase 2 SK180 H24-141GAGTCCAGCCTTC 140 cytochrome P450, HUMCYPIIF- J02906- ATGCCC subfamilyIIF, poly- CYP2F1 NM_000774 peptide 1 H24-142 GTCCAGCTGAAG 141 glutamatereceptor, GRIN2A NM_000833 AAGATCC ionotropic, N-methyl D- aspartate 2AH24-143 TTCGGCACTGAGG 142 hypothetical protein FLJ22955 NM_024819 TCTTGCFLJ22955 H24-145 CCTGCTCTTGAGC 144 tumor endothelial marker TEM5NM_032777 AATAAC 5 precursor H24-146 TGTCCAGACCACA 145 similar tocytochrome LOC126538 XM_065152 TGGAGC P450, subfamily IVF, polypeptide2; leuko- triene B4 omega-hydroxyl- ase; leukotriene-B4 20-monooxygenase H24-148 GATTGTGGCCAAG 147 chloride intracellular CLIC6XM_092804 AAGTAC channel 6 H24-149 CCTCATTATCACC 148 LOC167417 LOC167417XM_094471 ATGCTC H24-150 CTGGTTATTGGCG 149 similar to 25- LOC165245XM_103864 GGTATC hydroxyvitamin D-1 alpha hydroxylase, mitochondrialprecursor (25-OHD-1 alpha- hydroxylase) (25- hydroxyvitamin D3 1-alpha-hydroxylase) (VD3 1A hydroxylase) (P450C1 alpha) (P450VD1-alpha)H24-154 GTTCAAGAAGCTG 153 opsin 1 (cone pigments), OPN1MW- NM_000513-CGCCAC medium-wave-sensitive OPN1LW NM_020061 (color blindness,deutan)-opsin 1 (cone pigments), long-wave- sensitive (color blindness,protan) H24-156 GCAGTTCCAAGCT 155 opsin 1 (cone pigments), OPN1SWNM_001708 TGCATC short-wave-sensitive (color blindness, tritan) H24-157GTACCTGCGCCAC 156 chemokine (C—C motif) CCR3 NM_001837 TTCTTC receptor 3H24-159 GTCCTTCTACATC 158 G protein-coupled GPR23 NM_005296 AATGCCreceptor 23 H24-160 GAAGAAGCAACT 159 G protein-coupled GPR64 NM_005756GGGAGCC receptor 64 H24-169 CAACCTGTTCATC 164 galanin receptor 2 GALR2NM_003857 CTTAAC H24-173 GTTCTCTCAGCAC 168 placental growth factor, PGFNM_002632 GTTCGC vascular endothelial growth factor-related proteinH24-180 ATGCAGGTCAGGT 175 solute carrier family 4, SLC4A10 NM_022058TGTTTC sodium bicarbonate transporter-like, member 10 H24-185ACCGTGGAAGGC 180 cytochrome P450, CYP24 NM_000782 CTATCGC subfamily XXIV(vitamin D 24-hydroxylase) H24-188 TCGGCAGGGCCA 183 macrophagestimulating 1 MST1 NM_020998 GCATTTC (hepatocyte growth factor- like)H24-190 TCAGAAGGTTGTG 185 KIAA0943 protein Apg4B NM_013325 CAGGACH24-191 CAACTTGCATGAC 186 amyloid beta (A4) APP NM_000484 TACGGCprecursor protein (protease nexin-II, Alzheimer disease) H24-193ACCAGTGGTAAAT 188 dual specificity DUSP5 NM_004419 GTCAGC phosphatase 5H24-194 CTCTGTATCCCAT 189 mitogen-activated protein MAP3K9 XM_027237TCCCTC kinase kinase kinase 9 H24-200 GTAGCACTCTGCG 195 solute carrierfamily 39 SLC39A4 NM_017767- ACATGC (zinc transporter), NM_130849 member4 H24-202 GTTATTCTTCCAC 197 nicotinamide N- NNMT NM_006169 CATGGCmethyltransferase H24-205 AGCATGACAGGA 200 UDP-glucose ceramide UGCGL2NM_020121 AACCTGC glucosyltransferase- like 2 H24-207 CCTTGTTGGCCAA 202similar to arylacetamide LOC166161 XM_093702 TGATTC deacetylase(esterase) H24-211 CAAGTTCTCCTGC 206 ATPase, H+/K+ ATP4B NM_000705AAGTTC exchanging, beta polypeptide H24-218 CAACATCCCAACT 213glutathione reductase GSR NM_000637 GTGGTC H24-219 TATCCTGACCTTC 214potassium voltage-gated KCNG1 NM_002237 CTGCGC channel, subfamily G,member 1 H24-224 TATTCGTGCGGAG 219 chromosome 9 open SgK071 SK521 GAAGACreading frame 96 H24-225 ATGGGCTTCAACA 220 arginine vasopressin AVPR1BNM_000707 GCCACC receptor 1B

EXAMPLE 7 Drug Discovery Against the Identified TARGETs

Compounds are screened for binding to the polypeptides of the presentinvention. The affinity of the compounds to the polypeptides isdetermined in a displacement experiment. Such displacement experimentsare well known in the art, and can be considered as a common techniqueamong others to identify compounds that bind to polypeptides.

In brief, the polypeptides of the present invention are incubated with alabeled (radio-labeled, fluorescent- or antibody-labeled, or any otherdetectable label) ligand that is known to bind to the polypeptide and isfurther incubated with an unlabeled compound.

The displacement of the labeled ligand from the polypeptide isdetermined by measuring the amount of labeled ligand that is stillassociated with the polypeptide. The amount of the labeled ligandassociated with the peptide is an indication of the affinity for theunlabeled compound.

The amount of labeled ligand associated with the polypeptide is plottedagainst the concentration of the unlabeled compound to calculate IC50values. This value reflects the binding affinity of the unlabeledcompound to its TARGET, i.e. the polypeptides of the present invention.

Compounds are considered strong binders, when having an IC50 in thenanomolar and even picomolar range. Compounds that have an IC50 of atleast 10 micromol or even better in the nmol to pmol range are appliedin either the bone alkaline phosphatase assay (BAP) and/or in assays todetermine their effect on the induction of osteoblast markers andosteoblast function. Compounds with a lower IC50 are generallyconsidered as of less interest. The polypeptides of the presentinvention can be prepared in a number of ways depending on whether theassay will be run on cells, cell fractions, or biochemically on purifiedproteins. Such preparations are well known in the art, as are thedifferent assays.

EXAMPLE 8 Osteoclast Assays: Validate Anti-resorptive Activity ofIdentified TARGETs

Throughout life, the skeleton is in a constant state of remodeling.Focal areas of bone are resorbed by osteoclasts and then replaced bybone matrix newly formed by osteoblasts. The development of osteoporosisis characterized by severe bone loss due to the deregulation of thebalance between osteoclast and osteoblast activity, leading to anincreased osteoclast-mediated bone resorption.

Osteoclasts emanate from cells of the monocyte/macrophage lineage. Invivo, the differentiation of osteoclast precursor cells towardsosteoclasts is controlled by two central factors expressed by stromalcells (MPCs): receptor activator of NFκB ligand (RANKL) andosteoprotegerin (OPG). RANKL is a membrane bound ligand expressed on thesurface of MPCs which drives osteoclast differentiation. OPG is asoluble decoy receptor for RANKL which inhibits osteoclastdifferentiation by scavenging active RANKL. The balance between RANKLand OPG expression by MPCs determines the level of osteoclastdifferentiation.

As MPCs control the differentiation of osteoclasts, it is important toknow the effect of the identified TARGET Ad-siRNAs on osteoclastdifferentiation or activity. Target Ad-siRNAs that decrease osteoclastdifferentiation/activity, are very valuable, as these are expected toincrease bone apposition by two mechanisms: increase ofdifferentiation/activity of osteoblasts and decrease in osteoclastactivity. As illustrated by various precedents (Thirunavukkarasu et al.,(2000) J Biol Chem 275 : 25163-72 ; Yamada et al., (2003) Blood 101:2227-34) such a pleiotropic effect of osteogenic factors can beexpected.

Osteoclast Differentiation Assay

The effect of osteogenic factors on osteoclastogenesis is evaluatedthrough two types of assays.

In a first assay setup, a coculture of MPCs with primary humanmononuclear cells is performed. The effect of the infection of the MPCmonolayer with a knock-down virus on its capacity to supportosteoclastogenesis is evaluated. The desired effect is the following:knock-down of the Ad-siRNA TARGET gene expression in the MPCs shouldinhibit osteoclast differentiation driven by a physiological trigger ase.g. a mixture of 10 nM 1,25(OH)₂vitD₃ and 50 nM M-CSF. The monocytesused can be derived from bone marrow or peripheral blood. In the presentexample, a differentiation experiment based on peripheral blood derivedmononuclear cells (PBMCs) is described. MPCs (obtained fromCambrex/Biowhittaker, Verviers, Belgium) are seeded in 96 well plates(1000 cells per well) in α-MEM medium (GIBCO-Life Technologies)supplemented with 10% FBS and a day later, these are infected with aTARGET Ad-siRNA. At least three days later, 100 000 PBMCs per well areadded as well as M-CSF (R&D systems, 50 ng/ml final concentration). Halfthe volume of medium is refreshed twice a week by medium +50 ng/ml M-CSFand 10 nM 1,25(OH)₂vitD₃. Readout is performed 14 days after addition ofthe PBMCs to the coculture. Spontaneous osteoclast differentiationdriven by the physiologically relevant mixture of triggers can beassessed by multiple readouts. Microscopic assessment of the number of‘TRAP positive’, multinucleated cells per well is a generally acceptedmeasure for the level of osteoclast differentiation. ‘TRAP positive’means that the cells possess a tartrate resistant acidic phosphatase(TRAP) activity. To assess this, the coculture is subjected to an insitu TRAP staining performed according to the Acid Phosphatase detectionkit (SIGMA, 386-A). Positive cells aquire a purple color upon treatment.As an alternative readout, a marker specific for mature osteoclasts ismeasured e.g. TRACP5b (tartrate resistant acidic phosphatase type 5b),calcitonin receptor (CTR) or Cathepsin K (CTSK). Measurement of theamounts of osteoclast-derived tartrate resistant acidic phosphataseprotein (TRACP5b) in the coculture supernatant is performed by acommercially available ELISA (BoneTRAP assay, Sba sciences, Turku,Finland). CTR or CTSK are detected by immunocytochemistry, uponapplication of following general protocol. Medium is removed and thecoculture is fixed (4% paraformaldehyde, 0.1% TritonX-100, 4° C., 30min), washed and blocking buffer (PBS+1% BSA+0.1% Tween20) is added foran incubation of at least 4hrs. The blocking buffer is removed and theprimary antibody directed against CathepsinK (e.g. Oncogene, IM55L) orCalcitonin receptor (e.g. Serotec, AHP635), dissolved at the desiredconcentration in a suited buffer (e.g. 0.05M Tris.HCl pH 7.4, 1% BSA),is added to the wells. Incubation is performed overnight, 4° C. Themixture is removed, the cells washed (PBS+0.1% Tween20) and the suited,HRP conjugated secondary antibody, diluted in the same buffer as theprimary antibody, is added. After an incubation of at least 4 hrs, awashing step is performed (PBS+0.1% Tween20) and luminol (a substratefor HRP yielding a luminescent signal: BM Chemiluminescence ELISASubstrate [POD] (luminol), Roche Diagnostics, Cat No 1582950) is added.After 5 min incubation, readout is performed with a luminometer(Luminoskan Ascent, Labsystem). The 2 assays described (assessment ofthe amount of multinuclear cells and imnmunochemistry for the detectionof osteoclast-specific markers) allow to assess the differentiation ofthe mononuclear cells towards osteoclasts, but do not yield informationabout the bone resorptive activity of the osteoclasts formed.

Activity of the osteoclasts is measured in the pit formation assay. Forthis purpose, the co-culture and infection of cells is performed asdescribed for assays described above with the difference that abone-like substrate is present at the bottom of the well in which theco-culture is performed. This bone-like substrate can be a dentin slice(e.g. Kamiya Biomedical Company, Seattle (Cat No KT018)) or equivalent(Calcium carbonate coating, OAAS™, Gentaur; Biocoat™ Osteologic™, BDBiosciences) that is commercially available. The co-culture is performedfor at least 14 days on the bone like substrate. Cells are then removedby treatment with sodium hypochlorite and the area resorbed by theosteoclasts (the resorption pit) can be assessed microsopically. Thiscan be facilitated by the treatment of the surface of the dentin slicewith toluidine blue.

In a second assay setup, the effect of the infection of the osteoclastprecursor cells (PBMCs or BMMCs) with a TARGET virus on its ability todifferentiate towards an osteoclast is measured in a monoculture assay.For this purpose, the monocytes (PBMCs or BMMCs) are seeded in a 384well plate in aMEM medium supplemented with 10% serum and 25 ng/mlrecombinant M-CSF (R&D systems). One day after seeding, the cells areinfected with TARGET Ad-siRNAs. Four days after infection, recombinantRANKL is added to the wells (25 ng/ml, R&D systems). Medium is refreshedtwice a week. Fourteen days after addition of RANKL, the differentiationof the monocytes towards osteoclasts is measured using one of thereadouts described for the former assay setup. This assay allows theidentification of factors that are indispensable for the response ofosteoclast precursor cells to M-CSF or RANKL.

PBMC Isolation

PBMCs are obtained from peripheral blood (obtained from patients afterinformed consent) subjected to the following protocol. Blood isaseptically poured into 50 ml Falcon tubes and spun at 3000 g for 10 minat 25° C. The buffy coat is then collected and diluted 1:1 with PBS. Thediluted buffy coat is poured on top of 20 ml Lymphoprep (Sigma)contained in a 50 ml Falcon tube. Upon centrifugation (35 min at 400 gat 25° C.), a white layer of mononuclear cells on top of the Lymphoprepis collected and washed twice with PBS (centrifugation at 200 g, 10 min,25° C.) and rediluted in 7 ml PBS. This solution is pipetted onto alayer of 7 ml of hyperosmolar Percoll gradient contained in a 15 mlFalcon tube and centrifuged 35 min at 400 g at 25° C. The hyperosmolarPercoll gradient is prepared as follows: 1 volume of 1.5 M NaCl and 9volumes of Percoll (Pharmacia, d=1,130 g/ml) are mixed. This mixture isadded 1:1 to a PBS/Citrate buffer (NaH2PO4 1.49 mM, Na2HPO4 9.15 mM,NaCl 139.97 mM, Na-citrate (dihydrate) 13 mM, pH 7.2). Aftercentrifugation, monocytes form a discrete ring on top of the gradient.Monocytes are collected and washed in culture medium. Cells are thenready to use in assays.

EXAMPLE 9 Analysis of ‘off-target’ Knock Down Effect

SiRNAs exert knock-down of gene expression through a recently discoveredand partially understood mechanism. It is generally accepted that thespecific annealing of the siRNA sequence to mRNA is responsible for agene-specific ‘on-target’ knock-down. However, it cannot be excluded yetthat limited mismatching between the siRNA and another mRNA can induce‘off-target’ down-regulation of gene expression.

In order to exclude that the knock-down of (an) ‘off-target’ mRNA(s) wasresponsible for the observed osteogenic effect, additional siRNAs/shRNAsare designed for 38 targets that induced mineralization (Example 6)using stringent design criteria. The additional Ad-shRNAs are thentested in the BAP assay.

To address the question of possible ‘off-target’ effects, additionalsiRNA sequences are designed that align perfectly with the mRNA targetedby the original siRNA. Preferred siRNA sequences do not align to othermRNAs. However, in some cases only siRNAs could be designed that showedsome overlap to other mRNAs. For siRNAs that aligned to a minimal numberof ‘off-target’ mRNAs (maximum of 2 basepairs non-identity checked forevery position of the 19mer) the following rules are applied: theputative ‘off-target’ mRNAs must be different from the putative‘off-target’ mRNAs identified for the original siRNA; and putative‘off-target’ mRNAs must be different from the putative ‘off-target’mRNAs identified for all original target siRNAs. The only exception tothese rules made during the course of these experiments are siRNAsdesigned for PPIA.

For each of the 38 selected target genes, 7 additional siRNAs weredesigned and processed to derive recombinant adenoviruses. All siRNAsare sequenced upon cloning, to verify their identity and exclude errorsdue to the oligonucleotide synthesis.

261 Ad-shRNAS were successfully generated and tested in the BAP assay at3 MOIs in 2 independent experiments, in parallel with the original 38Ad-shRNAs.

Recombinant adenoviruses encoding the designed shRNAs (Ad-shRNAs) areproduced, titered, aliquoted in 96 well plates and stored at −80° C.These plates were processed in the primary BAP assay as follows:

MPC cells wearere seeded with a Multidrop 384 (Labsystems) in black 384well plates with clear bottom (Costar or Nunc) in 60 μl MSC mediumcontaining 10% fetal calf serum (Progentix, The Netherlands), at adensity of 500 cells per well. One day later, a 96 well plate containingaliquoted Ad-shRNAs and another containing negative and positive controlviruses (knock-down control plate) are thawed and virus aliquotstransferred to the MPC plate using a 96-channel dispenser (Tecan Freedom200 equipped with a TeMO96 and a RoMa plate handler, Tecan AG,Switzerland). For the control plate, 1 μL virus stock (average titer of2×10⁹ viral particles per ml) is transferred to the 384 well screeningplates. On the control plate (see FIG. 3), negative (N1, N2, N3) andpositive (P1, P2) control viruses are diagonally distributed over theplate. N1, N2, N3: Ad-siRNAs targeting the eGFP,mannose-6-phosphate-receptor and luciferase mRNAs, respectively. P1 andP2: Ad-siRNAs targeting the PRKCN (H24-010) and MPP6 (H24-011) mRNAs.P3: Ad-eGFP: overexpression of eGFP allows monitoring of infectionefficiency.

The Ad-shRNAs are screened at 3 multiplicities of infection (MOIs):12,000, 4,000 and 1,333. Ad-shRNAs are aliquotted into the inner wellsof a 96 well plate at a titer of 1.5×10⁹/4 μl and 1/3 and 1/9 dilutionsare derived of this plate (FIG. 11). The resulting 3 plates are used toinfect 3×384 well plates seeded with MSCs, in the A1 and B2 positions ofeach quadrant using robotics. The viruses from the control plate arepipetted in the A2 and B1 positions.

Next, 5 μl of adenovirus expressing the human coxsackie and adenovirusreceptor (hCAR) (AdC15-hCAR/AdC20-hCAR) is transferred into these wells(final MOI of 155) from a 96 well V-bottom plate with the aid of the96-channel dispenser.

Plates are then incubated at 37° C., 10% CO₂ in a humidified incubatorfor four days. Four days post infection, the medium containing theadenoviruses is replaced by 60 μl fresh MSC medium containing 10% FCSfree of virus. After an additional nine days of incubation, medium isremoved, 15 μL of a 4-methylumbelliferylphosphate solution (Sigma,#M3168) is added to each well and the fluorescence of4-methyl-umbelliferone released by the alkaline phosphatase activity ismeasured after 15 min incubation at 37° C. using a fluorimeter(excitation: 360 nm; emission: 440 nm; FluoStar, BMG).

All Ad-shRNAs viruses are screened in duplicate at 3 MOIs in twoindependent screens. Thresholds are calculated for hit calling usingeither all negative controls present in one screening round (‘Global’analysis) or using the negative controls present on one screening plate(‘Local’ analysis). Threshold is set at a BAP signal higher than themean plus 3 times the standard deviation of negative controls. The twoindividual datapoints for each virus in the batch are analyzedindependently. Hits are selected if one Ad-shRNAs scored at least at oneMOI in duplicate in at least one of the 2 screens above the threshold(see Table 7).

A ‘Global’ analysis of the data identified 61 siRNAs targeting 32 lociand a ‘Local’ analysis' identified 84 siRNAs targeting 35 loci. Theidentity of the 38 selected genes is presented in Table 7 together withthe final number of siRNAs that scored in the BAP assay. In this Table,the numbers indicate all siRNAs that scored in the BAP assay, includingthe original 38 siRNAs. Based on the ‘Global’ and ‘Local’ analysis, anaverage of 2.61 and 3.21 constructs respectively are identified for eachof the 38 validated targets.

All original 38 Ad-shRNAs scored in the BAP assay based on both the‘Global’ and ‘Local’ analysis.

In conclusion, additional Ad-shRNAs targeting 38 selected targets aredesigned and constructed. Negative controls present on the controlplates are used per plate (‘Local’ analysis) or per batch of plates(‘Global’ analysis) to determine the cutoff for hit calling.

The ‘Global’ analysis results in 61 viruses that scored positive in theBAP assay, confirming 32 of the 38 validated targets. The ‘Local’analysis results in 84 viruses that scored positive in the BAP assay,confirming 35 of the 38 validated targets. All original 38 Ad-shRNAviruses score in the BAP assay when using either the ‘Global’ or the‘Local’ analysis. The targets LOC160662, PPIA and SLC39A4 are identifiedonly in the ‘Local’ analysis.

TABLE 7 TARGET Gene Global analysis - Local analysis - Symbol RedundancyRedundancy APG4B 2 3 C13orf6 2 2 C20orf121 3 4 CCR1 3 4 CCR3 2 2 CEL 2 4CLIC6 2 2 CSNK1G1 2 3 DUSP5 2 2 FLJ22955 3 4 FRK 3 3 GPR124 2 3 GPR23 23 GPR64 4 5 Grin2a-GRIN2A 7 6 GZMK 2 2 HRMT1L3 2 2 IRAK2 4 4 LOC160662 -LST-3 1 2 LOC167417 - GPR150 4 5 LOC254378 3 4 MAP3K9 4 5 MLNR 4 3MMP23B-MMP23A 3 2 MNK2 2 3 OR1A2 4 6 PPIA 1 3 RASD1 4 5 RDH11 3 4 SENP34 4 SLC16A3 2 3 SLC26A8 1 1 SLC39A4 1 2 SLC4A10 1 1 TAO1 3 4 TAS1R3 2 3TRPM6 1 1 ULK1 2 3

EXAMPLE 10 Identification of Small Molecules that Inhibit TARGET KinaseActivity

Compounds are screened for inhibition of the activity of the TARGETSthat are kinase polypeptides. The affinity of the compounds to thepolypeptides is determined in an experiment detecting changed reactionconditions after phosphorylation. The TARGET kinase polypeptides areincubated with its substrate and ATP in an appropriate buffer. Thecombination of these components results in the in vitro phosphorylationof the substrate. Sources of compounds include commercially availablescreening library, peptides in a phage display library or an antibodyfragment library, and compounds that have been demonstrated to havebinding affinity for a TARGET kinase.

The TARGET kinase polypeptides can be prepared in a number of waysdepending on whether the assay will be run using cells, cell fractionsor biochemically, on purified proteins. The polypeptides can be appliedas complete polypeptides or as polypeptide fragments, which stillcomprise TARGET kinase catalytic activity.

Identification of small molecules inhibiting the activity of the TARGETkinase polypeptides is performed by measuring changes in levels ofphosphorylated substrate or ATP. Since ATP is consumed during thephosphorylation of the substrate, its levels correlate with the kinaseactivity. Measuring ATP levels via chemiluminescent reactions thereforerepresents a method to measure kinase activity in vitro (Perkin Elmer).In a second type of assay, changes in the levels of phosphorylatedsubstrate are detected with phosphospecific agents and are correlated tokinase activity. These levels are detected in solution or afterimmobilization of the substrate on a microtiter plate or other carrier.In solution, the phosphorylated substrate is detected via fluorescenceresonance energy transfer (FRET) between the Eu labeled substrate and anAPC labeled phosphospecific antibody (Perkin Elmer), via fluorescencepolarization (FP) after binding of a phosphospecific antibody to thefluorescently labeled phosphorylated substrate (Panvera), via anAmplified Luminescent Proximity Homogeneous Assay (ALPHA) using thephosphorylated substrate and phosphospecific antibody, both coupled toALPHA beads (Perkin Elmer) or using the IMAP binding reagent thatspecifically detects phosphate groups and thus alleviates the use of thephosphospecific antibody (Molecular Devices). Alternatively, thesubstrate is immobilized directly or by using biotin-streptavidin on amicrotiter plate. After immobilization, the level of phosphorylatedsubstrate is detected using a classical ELISA where binding of thephosphospecific antibody is either monitored via an enzyme such ashorseradish peroxidase (HRP) or alkaline phospahtase (AP) which areeither directly coupled to the phosphospecific antibody or are coupledto a secondary antibody. Enzymatic activity correlates to phosphorylatedsubstrate levels. Alternatively, binding of the Eu-labeledphosphospecific antibody to the immobilized phosphorylated substrate isdetermined via time resolved fluorescence energy (TRF) (Perkin Elmer).In addition, the substrate can be coated on FLASH plates (Perkin Elmer)and phosphorylation of the substrate is detected using ³³P labeled ATPor ¹²⁵I labeled phosphospecific antibody.

Small molecules are randomly screened or are preselected based upon drugclass, (i.e. known kinase inhibitors), or upon virtual ligand screening(VLS) results. VLS uses virtual docking technology to test large numbersof small molecules in silico for their binding to the polypeptide of theinvention. Small molecules are added to the kinase reaction and theireffect on levels of phosphorylated substrate is measured with one ormore of the above-described technologies.

Small molecules that inhibit the kinase activity are identified and aresubsequently tested at different concentrations. IC₅₀ values arecalculated from these dose response curves. Strong binders have an IC₅₀in the nanomolar and even picomolar range. Compounds that have an IC₅₀of at least 10 micromol or better (nmol to pmol) are applied in alkalinephosphatase assay or bone mineralization assay to check for their effecton the induction of osteogenesis.

EXAMPLE 11 Ligand Screens for TARGET GPCRs

Reporter Gene Screen.

Mammalian cells such as Hek293 or CHO-K1 cells are either stablytransfected with a plasmid harboring the luciferase gene under thecontrol of a cAMP dependent promoter (CRE elements) or transduced withan adenovirus harboring a luciferase gene under the control of a cAMPdependent promoter. In addition reporter constructs can be used with theluciferase gene under the control of a Ca²⁺ dependent promoter (NF-ATelements) or a promoter that is controlled by activated NF-κB. Thesecells, expressing the reporter construct, are then transduced with anadenovirus harboring the cDNA of a TARGET GPCR. Forty (40) hours aftertransduction the cells are treated with the following:

a) an agonist for the receptor and screened against a large collectionof reference compounds comprising peptides (LOPAP, Sigma Aldrich),lipids (Biomol, TimTech), carbohydrates (Specs), natural compounds(Specs, TimTech), small chemical compounds (Tocris), commerciallyavailable screening libraries, and compounds that have been demonstratedto have binding affinity for a polypeptide comprising an amino acidsequence selected from the group consisting of the SEQ ID NOs of theTARGET GPCRs; or

b) a large collection of reference compounds comprising peptides (LOPAP,Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), naturalcompounds (Specs, TimTech), small chemical compounds (Tocris),commercially available screening libraries, and compounds that have beendemonstrated to have binding affinity for a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs ofthe TARGET GPCRs.

Compounds, which decrease the agonist induced increase in luciferaseactivity or the constitutive activity, are considered to be antagonistsor inverse agonists for a TARGET GPCR. These compounds are screenedagain for verification and screened against their effect on osteoblastdifferentiation. The compounds are also screened to verify binding tothe GPCR. The binding, osteogenesis and reporter activity assays can beperformed in essentially any order to screen compounds.

In addition, cells expressing the NF-AT reporter gene can be transducedwith an adenovirus harboring the cDNA encoding the α-subunit of G₁₅ orchimerical Gα subunits. G₁₅ is a promiscuous G protein of the G_(q)class that couples to many different GPCRs and as such re-directs theirsignaling towards the release of intracellular Ca²⁺ stores. Thechimerical G alpha subunits are members of the G_(s) and G_(i/o) familyby which the last 5 C-terminal residues are replaced by those of G_(αq),these chimerical G-proteins also redirect cAMP signaling to Ca²⁺signaling.

FLIPR Screen.

Mammalian cells such as Hek293 or CHO-K1 cells are stably transfectedwith an expression plasmid construct harboring the cDNA of a TARGETGPCR. Cells are seeded, grown, and selected until sufficient stablecells can be obtained. Cells are loaded with a Ca²⁺ dependentfluorophore such as Fura3 or Fura4. After washing away the excess offluorophore the cells are screened against a large collection ofreference compounds comprising peptides (LOPAP, Sigma Aldrich), lipids(Biomol, TimTech), carbohydrates (Specs), natural compounds (Specs,TimTech), small chemical compounds (Tocris), commercially availablescreening libraries, and compounds that have been demonstrated to havebinding affinity for a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs of the TARGET GPCRs, bysimultaneously adding an agonist (alternatively no agonist need be addedif the constitutive activity of the receptor is used) and a compound tothe cells. Activation of the receptor is measured as an almostinstantaneously increase in fluorescence due to the interaction of thefluorophore and the Ca²⁺ that is released. Compounds that reduce orinhibit the agonist induced increase in fluorescence (or constitutivefluorescence) are considered to be antagonists or inverse agonists forthe receptor they are screened against. These compounds are screenedagain to measure the amount of osteoblast differentiation as well asbinding to a TARGET GPCR.

AequoScreen.

CHO cells, stably expressing Apoaequorin are stably transfected with aplasmid construct harboring the cDNA of a TARGET GPCR. Cells are seeded,grown, and selected until sufficient stable cells can be obtained. Thecells are loaded with coelenterazine, a cofactor for apoaequorin. Uponreceptor activation intracellular Ca²⁺ stores are emptied and theaequorin will react with the coelenterazine in a light emitting process.The emitted light is a measure for receptor activation. The CHO, stableexpressing both the apoaequorin and the receptor are screened against alarge collection of reference compounds comprising peptides (LOPAP,Sigma Aldrich), lipids (Biomol, TimTech), carbohydrates (Specs), naturalcompounds (Specs, TimTech), small chemical compounds (Tocris),commercially available screening libraries, and compounds that have beendemonstrated to have binding affinity for a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs ofthe TARGET GPCRs, by simultaneously adding an agonist (alternatively noagonist need be added if the constitutive activity of the receptor isused) and a compound to the cells. Activation of the receptor ismeasured as an almost instantaneously light flash due to the interactionof the apoaequorin, coelenterazine, and the Ca²⁺ that is released.Compounds that reduce or inhibit the agonist induced increase in lightor the constitutive activity are considered to be antagonists or inverseagonists for the receptor they are screened against. These compounds arescreened again to measure the amount of osteoblast differentiation aswell as binding to a TARGET GPCR.

In addition, CHO cells stable expressing the apoaequorin gene are stablytransfected with a plasmid construct harboring the cDNA encoding theα-subunit of G15 or chimerical G_(α) subunits. G₁₅ is a promiscuous Gprotein of the G_(q) class that couples to many different GPCRs and assuch redirects their signaling towards the release of intracellular Ca²⁺stores. The chimerical G alpha subunits are members of the G_(s) andG_(i/o) family by which the last 5 C-terminal residues are replaced bythose of G_(αq), these chimerical G-proteins also redirect cAMPsignaling to Ca²⁺ signaling.

Screening for Compounds that Bind to the GPCR Polypeptides (DisplacementExperiment)

Compounds are screened for binding to the TARGET GPCR polypeptides. Theaffinity of the compounds to the polypeptides is determined in adisplacement experiment. In brief, the GPCR polypeptides are incubatedwith a labeled (radiolabeled, fluorescent labeled) ligand that is knownto bind to the polypeptide and with an unlabeled compound. Thedisplacement of the labeled ligand from the polypeptide is determined bymeasuring the amount of labeled ligand that is still associated with thepolypeptide. The amount associated with the polypeptide is plottedagainst the concentration of the compound to calculate IC₅₀ values. Thisvalue reflects the binding affinity of the compound to its TARGET, i.e.the TARGET GPCR polypeptides. Strong binders have an IC₅₀ in thenanomolar and even picomolar range. Compounds that have an IC₅₀ of atleast 10 micromol or better (nmol to pmol) are applied an osteoblastdifferentiation assay to check for their effect on osteogenesis. TheTARGET GPCR polypeptides can be prepared in a number of ways dependingon whether the assay are run on cells, cell fractions or biochemically,on purified proteins. Screening for compounds that bind to a TARGET GPCR(generic GPCR screening assay) When a G protein receptor becomesconstitutively active, it binds to a G protein (G_(q), G_(s), G_(i),G_(o)) and stimulates the binding of GTP to the G protein. The G proteinthen acts as a GTPase and slowly hydrolyses the GTP to GDP, whereby thereceptor, under normal conditions, becomes deactivated. However,constitutively activated receptors continue to exchange GDP to GTP. Anon-hydrolyzable analog of GTP, [³⁵S]GTPγS, can be used to monitorenhanced binding to membranes which express constitutively activatedreceptors. It is reported that [³⁵S]GTPγS can be used to monitor Gprotein coupling to membranes in the absence and presence of ligand.Moreover, a preferred approach is the use of a GPCR-G protein fusionprotein. The strategy to generate a TARGET GPCR-G protein fusion proteinis well known for those known in the art. Membranes expressing TARGETGPCR-G protein fusion protein are prepared for use in the directidentification of candidate compounds such as inverse agonist.Homogenized membranes with TARGET GPCR-G protein fusion protein aretransferred in a 96-well plate. A pin-tool is used to transfer acandidate compound in each well plus [³⁵S]GTPγS, followed by incubationon a shaker for 60 minutes at room temperature. The assay is stopped byspinning of the plates at 4000 RPM for 15 minutes at 22° C. The platesare then aspirated and radioactivity is then read.

Receptor Ligand Binding Study on Cell Surface

The receptor is expressed in mammalian cells (Hek293, CHO, COS7) byadenoviral transducing the cells (see U.S. Pat. No. 6,340,595). Thecells are incubated with both labeled ligand (iodinated, tritiated, orfluorescent) and the unlabeled compound at various concentrations,ranging from 10 pM to 10 μM (3 hours at 4° C.: 25 mM HEPES, 140 mM NaCl,1 mM CaCl₂, 5 mM MgCl₂ and 0.2% BSA, adjusted to pH 7.4). Reactionsmixtures are aspirated onto PEI-treated GF/B glass filters using a cellharvester (Packard). The filters are washed twice with ice cold washbuffer (25 mM HEPES, 500 mM NaCl, 1 mM CaCl₂, 5 mM MgCl₂, adjusted to pH7.4). Scintillant (MicroScint-10; 35 μl) is added to dried filters andthe filters counted in a (Packard Topcount) scintillation counter. Dataare analyzed and plotted using Prism software (GraphPad Software, SanDiego, Calif.). Competition curves are analyzed and IC₅₀ valuescalculated. If one or more data points do not fall within the sigmoidalrange of the competition curve or close to the sigmoidal range the assayis repeated and concentrations of labeled ligand and unlabeled compoundadapted to have more data points close to or in the sigmoidal range ofthe curve.

Receptor Ligand Binding Studies on Membrane Preparations

Membranes preparations are isolated from mammalian cells (Hek293, CHO,COS7) cells over expressing the receptor is done as follows: Medium isaspirated from the transduced cells and cells are harvested in 1× PBS bygentle scraping. Cells are pelleted (2500 rpm 5 min) and resuspended in50 mM Tris pH 7.4 (10×10⁶ cells/ml). The cell pellet is homogenized bysonicating 3×5 sec (UP50H; sonotrode MS1; max amplitude: 140 μm; maxSonic Power Density: 125W/cm²). Membrane fractions are prepared bycentrifuging 20 min at maximal speed (13,000 rpm ˜15,000 to 20,000g orrcf). The resulting pellet is resuspended in 500 μl 50 mM Tris pH 7.4and sonicated again for 3×5 sec. The membrane fraction is isolated bycentrifugation and finally resuspended in PBS. Binding competition andderivation of IC₅₀ values are determined as described above.

Internalization Screen (1)

Activation of a GPCR-associated signal transduction pathway commonlyleads to translocation of specific signal transduction molecules fromthe cytoplasm to the plasma membrane or from the cytoplasm to thenucleus. Norak has developed their transfluor assay based onagonist-induced translocation of receptor-β-arrestin-GFP complex fromthe cytosol to the plasma membrane and subsequent internalization ofthis complex, which occurs during receptor desensitization. A similarassay uses GFP tagged receptor instead of β-arrestin. Hek293 cells aretransduced with a TARGET GPCR vector that translates for a TARGETGPCR-EGFP fusion protein. 48 hours after transduction, the cells are setto fresh serum-free medium for 60 minutes and treated with a ligand for15, 30, 60 or 120 minutes at 37° C. and 5% CO₂. After indicated exposuretimes, cells are washed with PBS and fixed with 5% paraformaldehyde for20 minutes at RT. GFP fluorescence is visualized with a Zeiss microscopewith a digital camera. This method aims for the identification ofcompounds that inhibit a ligand-mediated (constitutiveactivity-mediated) translocation of the fusion protein to intracellularcompartments.

Internalization Screen (2)

Various variations on translocation assays exists using β-arrestin andβ-galactosidase enzyme complementation and BRET based assays withreceptor as energy donor and β-arrestin as energy acceptor. Also the useof specific receptor antibodies labeled with pH sensitive dyes are usedto detect agonist induced receptor translocation to acidic lysosomes.All of the translocation assays are used for screening for bothagonistic and antagonistic acting ligands.

Melanophore Assay (Arena Pharmaceutical)

The melanophore assay is based on the ability of GPCRs to alter thedistribution of melanin containing melanosomes in Xenopus melanophores.The distribution of the melanosomes depends on the exogenous receptorthat is either G_(i/o) or G_(s/q) coupled. The distribution of themelanosomes (dispersed or aggregated) is easily detected by measuringlight absorption. This type of assay is used for both agonist as well asantagonist compound screens.

REFERENCES

-   Cortez-Retamozo et al. (2004) Cancer Res 64: 2853-7.-   Franceschi R T and Xiao G (2003) J Cell Biochem 8:446-454.-   Lipinsky, C A, et al. (2001) Adv Drug Deliv Rev 46: 3-26.-   Nakashima K, de Crombrugghe B. (2003) Trends Genet. 19(8):458-66.-   Marzia M, et al. (2000) J Cell Biol 151:311.-   Thirunavukkarasu et al., (2000) J Biol Chem 275: 25163-72.-   Yamada et al. (2003) Blood 101: 2227-34.-   Zhang et al. (2000) PNAS 97: 10549-10554.

1. A method for identifying a compound that induces differentiation ofundifferentiated vertebrate cells into osteoblasts, comprising (a)contacting a compound with a polypeptide consisting of an amino acidsequence of SEQ ID NO: 379, in an in vitro cell-free preparation; (b)measuring the binding affinity of said compound to said polypeptide; and(c) selecting a compound for confirmation as an inducer of vertebratecell differentiation into osteoblasts, which compound is selected basedon its binding affinity for the polypeptide of SEQ ID NO.
 379. 2. Amethod according to claim 1 for identifying a compound that inducesdifferentiation of undifferentiated mammalian vertebrate cells intoosteoblasts, said method further comprising c) contacting said compoundselected to have binding affinity to, and capable of forming a complexwith, said polypeptide of SEQ ID NO: 379 with a undifferentiatedmammalian vertebrate cell, which is in culture, and in which saidpolypeptide comprising the amino acid sequence of SEQ ID NO: 379 isexpressed; and (d) measuring in said culture levels of at least onebiochemical marker indicative of the differentiation of saidundifferentiated mammalian vertebrate cells; and (e) determining if saidlevels of said one or more biochemical marker indicative of thedifferentiation of said undifferentiated mammalian vertebrate cells aredecreased as compared to levels of said biochemical marker expressed insaid undifferentiated mammalian vertebrate cell that is not contactedwith said compound; and (f) selecting a compound, based on its decreasein biochemical marker level, for confirmation as an inducer ofvertebrate cell differentiation into osteoblasts.
 3. The methodaccording to claim 2, wherein said biochemical marker is bone alkalinephosphatase.
 4. The method according to claim 2 wherein said compoundhaving binding affinity to, and capable of forming a complex with, saidpolypeptide of SEQ ID NO: 379 exhibits a binding affinity of at least 10micromolar.
 5. The method according to claim 4, wherein saidundifferentiated mammalian vertebrate cell is an osteoblast progenitorcell.